Treatment of chemotherapeutic agent and antiviral agent toxicity with acylated pyrimidine nucleosides

ABSTRACT

The subject invention discloses compounds, compositions and methods for treatment and prevention of toxicity due to chemotherapeutic agents and antiviral agents. Disclosed are acylated derivatives of non-methylated pyrimidine nucleosides. These compounds are capable of attenuating damage to the hematopoietic system in animals receiving antiviral or antineoplastic chemotherapy.

This is a Divisional of application Ser. No. 08/176,485, filed Dec. 30,1993 now U.S. Pat. No. 5,736,531, which is a CIP of Ser. No. 08/061,381filed May 14, 1993 now abandoned, which is a CIP of Ser. No. 07/903,107filed Jun. 25, 1992 now abandoned, which is a CIP of Ser. No. 07/724,340filed Jul. 5, 1991 (abandoned), which is a CIP of Ser. No. 07/438,493filed Jun. 26, 1990 (abandoned), which is a CIP of Ser. No. 07/115,929filed Oct. 28, 1987 (abandoned), and Ser. No. 07/724,340 Jul. 5, 1991 inturn is a CIP of Ser. No. 07/487,984 filed Feb. 5, 1990 (abandoned),which is a CIP of Ser. No. 07/115,923 filed Oct. 28, 1987 (abandoned).All of these applications are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to treatment of chemotherapeutic agentand antiviral agent toxicity with acylated derivatives of non-methylatedpyrimidine nucleosides. These compounds are capable of attenuatingdamage to the hematopoietic system in animals receiving antiviral orantineoplastic chemotherapy. This invention also relates to protectionof other tissues affected by antiviral or antineoplastic chemotherapy,including the gastrointestinal epithelium.

BACKGROUND OF THE INVENTION

A major complication of cancer chemotherapy and of antiviralchemotherapy is damage to bone marrow cells or suppression of theirfunction. Specifically, chemotherapy damages or destroys hematopoieticprecursor cells, primarily found in the bone marrow and spleen,impairing the production of new blood cells (granulocytes, lymphocytes,erythrocytes, monocytes, platelets, etc.). Treatment of cancer patientswith 5-fluorouracil, for example, reduces the number of leukocytes(lymphocytes and/or granulocytes), and can result in enhancedsusceptibility of the patients to infection. Many cancer patients die ofinfection or other consequences of hematopoietic failure subsequent tochemotherapy. Chemotherapeutic agents can also result in subnormalformation of platelets which produces a propensity toward hemorrhage.Inhibition of erythrocyte production can result in anemia. The risk ofdamage to the hematopoietic system or other important tissues canprevent utilization of doses of chemotherapy agents high enough toprovide good antitumor or antiviral efficacy.

Many antineoplastic or antiviral chemotherapy agents act by inhibitingnucleotide biosynthesis, metabolism, or function, or are in factnucleoside analogs that substitute for the normal nucleosides in nucleicacids, producing defective RNA or DNA.

5-Fluorouracil is a clinically important cytoreductive antineoplasticchemotherapy agent that acts in part through incorporation into RNA,producing defective RNA; inhibition of thymidylate synthetase byfluorodeoxyuridine monophosphate may also contribute to the cytotoxicityof 5-FU. The clinical utility of 5-FU is limited by its toxicity(especially to bone marrow). Specifically, its clinical utility islimited by a low therapeutic ratio (the ratio of toxic dose to effectivedose; a high therapeutic ratio implies that a drug has efficacy withlittle toxicity).

5-FU and many other chemotherapy agents also affect other tissues,especially gastrointestinal mucosa, producing mucositis, diarrhea andulceration. Stomatitis (ulceration of mucosa in the mouth), isparticularly troublesome to patients, making eating and swallowingpainful.

D. S. Martin et al. (Cancer Res. 42:3964-70 [1982]) reported that atoxic dose of 5-FU (with strong anti-tumor activity) could be safelyadministered to mice if followed by administration of a high dose ofuridine beginning several hours later. This “rescue” strategy has beenshown to increase the therapeutic index of 5-FU in animal tumor models,allowing administration of the high, toxic doses of 5-FU that arenecessary for causing tumor regression or preventing tumor growth whilepreferentially protecting normal tissues (especially important is bonemarrow) by subsequent administration of uridine (D. S. Martin et al.,Cancer Res. 43:4653-61 [1983]).

Clinical-trials involving the administration of uridine have beencomplicated due to the biological properties of uridine itself. Uridineis poorly absorbed after oral administration; diarrhea is dose limitingin humans (van Groeningen et al., Proceedings of the AACR 28:195[1987]). Consequently, parenteral administration of uridine is necessaryfor clinically significant reversal of 5-FU toxicity, which requires useof a central venous catheter, since phlebitis has been a problem inearly clinical trials when uridine was administered via a smallintravenous catheter (van Groeningen et al. Cancer Treat Rep. 70:745-50[1986]). Prolonged infusion via central venous catheters requireshospitalization of the patients. Further, there is considerablediscomfort and inconvenience to the patients.

Orally-active prodrugs of 5FU have been developed which areenzymatically or spontaneously converted to SFU, generally afterabsorption from the intestine into the bloodstream. This permitsself-administration by patients, without the discomfort of intravenousadministration. Moreover, in some chemotherapy regimens, sustainedexposure, e.g. a constant intravenous infusion for several days orweeks, of tumors to 5FU is attempted. Oral administration of 5FUprodrugs can in principle provide such sustained availability of 5FU totumors.

5-Fluoro-1-(tetrahydro-2-furfuryl)uracil (FT) is an orally activeprodrug of 5-fluorouracil. It is enzymatically converted to5-fluorouracil primarily in the liver. The liver, however, hasrelatively high levels of the enzyme dihydropyrimidine dehydrogenase,which degrades 5FU, producing metabolites which are not useful in cancerchemotherapy and which furthermore contribute to 5-FU toxicity.

The cytotoxicity of 5FU, the active metabolite of FT, is believed to bea result of its incorporation into nucleotide pools, where certainanabolites exert toxic effects, e.g. 5-fluorodeoxyuridine monophosphateinhibits thymidylate synthetase, thus depriving cells of thymidine forDNA synthesis, and 5-fluorouridine triphosphate incorporation into RNAimpairs its normal functions in translation of genetic information.

In order to inhibit the catabolism of 5FU derived from FT, othercompounds have been administered with the FT. In particular, thepyrimidine uracil inhibits the catabolism of 5FU without inhibiting itscytotoxicity. The most widely used clinical formulation of FT containsuracil in a 1:4 molar ratio. This permits a significant reduction in thedose of FT required to achieve a therapeutic effect. Other pyridimines,including uridine, thymidine, thymine, and cytosine are either lesseffective than uracil or no better in potentiating the antitumorefficacy of FT without unacceptably potentiating toxicity. Potentsynthetic inhibitors of dihydropyrimidine dehydrogenase (DHPDHase) havealso been utilized with FT or 5FU. 5-chloro-2,6-dihydroxypyridine (CDHP)is more potent than uracil as an inhibitor of DHPDHase. However, thiscompound also enhances the toxicity of 5FU, so that, in its intendedclinical implementation, a third component, oxonic acid, isco-administered to reduce the intestinal toxicity.

Several investigators have administered pyrimidines with 5FU attemptingto improve the therapeutic index of this antineoplastic agent. In vivo,uridine and thymidine when administered at the same time as 5FUincreased both the antitumor efficacy of 5FU and its toxicity, so thatthere was no net increase in therapeutic index (Hartman and Bollag, Med.Oncol. & Tumor Pharmacother., 3:111-118 [1986]). Burchenal et al.(Cancer Chemother. Rep., 6:1-5 [1960]) summarized comprehensive studieson interactions of 5FU and 5-fluorodeoxyuridine (FUDR) and pyrimidinecompounds. They noted that despite the fact that pyrimidines andpyrimidine nucleosides, at doses which are inactive alone, markedlypotentiate the antileukemic effects of small doses of FUDR or FU, it hasnot been possible with any combination to improve significantly and withany degree of regularity the results which can be obtained with maximumtolerated doses of FU or FUDR alone. Similarly, Jato et al. (J. PharmSci., 64:943-945 [1975]), in an investigation of combinations ofdeoxyuridine with 5FU and FUDR report that any therapeutic benefit ofthe combination therapy could be duplicated with either 5FU or FUDR at ahigher dose. Although deoxyuridine, by inhibiting the catabolism of thefluoropyrimdines permitted administration of lower doses, deoxyuridinethere was no improvement in antitumor activity at equitoxic doses of thecombination versus FU or FUDR alone.

As in the case of uridine, problems of poor bioavailability after oraladministration limit the clinical utility of administration ofdeoxycytidine, cytidine, and deoxyuridine themselves for modulation oftoxicity of chemotherapy agents.

Arabinosyl cytosine (Ara-C) is an important agent in the treatment ofleukemia, and is also useful as an immunosuppressant. Bone marrowtoxicity (myeloid and erythroid) associated with Ara-C administrationcan be partially prevented by administration of deoxycytidine(Belyanchikova et al. Bull. Exp. Biol. Med. 91:83-85 [1981]), while thetoxicity of Ara-C to lymphocytes is not as strongly attenuated bydeoxycytidine. In cell cultures, normal myeloid progenitor cells areprotected from Ara-C by deoxycytidine better than are leukemic cells (K.Bhalla et al. Blood 70:568-571 [1987]). Deoxycytidine also attenuatestoxicity of 5-aza-2′-deoxycytidine and arabinosyl 5-azacytosine in cellcultures (K. Bhalla et al. Leukemia 1:814-819 [1987]). Prolonged (5 day)infusion of high doses of deoxycytidine via a central venous catheterwas proposed as a means for clinical implementation of modulation ofAra-C toxicity with deoxycytidine (K. Bhalla et al. Leukemia 2:709-710[1988]).

N-phosphonoacetyl-L-aspartic acid (PALA) is an antineoplastic agent thatinhibits the enzyme aspartate transcarbamoylase, an enzyme indirectlyinvolved in biosynthesis of pyrimidine nucleotides. Side effects of PALAprimarily involve damage to gastrointestinal toxicity and mucositis.Pyrazofurin (a carbon linked pyrimidine analog), 6-azauridine, and6-azacytidine all interfere with pyrimidine nucleotide synthesis andmetabolism.

3′-Azidodeoxythymidine (AZT) is used clinically, in patients infectedwith Human Immunodeficiency Virus (HIV, the infectious agent in AIDS).AZT prolongs the lifespan of patients infected with HIV, but alsoimpairs hematopoiesis, producing leukopenia and anemia. In cellcultures, uridine ameliorates AZT-induced toxicity togranulocyte/macrophage progenitor cells without impairing the antiviralactions of AZT (Sommadossi et al., (1988) Antimicrobial Agents andChemotherapy, 32:997-1001); thymidine attenuated both toxicity andantiviral activity. In mice, parenteral administration of high doses ofuridine provided some amelioration of AZT-induced anemia, but only aturidine doses which increased mortality during the study; a low,non-toxic dose of uridine (500 mg/kg/d) did not reduce AZT-inducedhematologic toxicity (A. Falcone, et al. Blood 76:2216-21 [1990]).Sommadossi and el Kouni (U.S. Pat. No. 5,077,280) proposed theadministration of uridine by periodic intravenous injection in order toattenuate AZT toxicity. Bhalla et al. (Blood 74:1923-1928 [1989])reported that deoxycytidine protects normal human bone marrow progenitorcells in vitro against the cytotoxicity of AZT with preservation ofantiretroviral activity.

5-Fluoroorotate, an analog of the pyrimidine nucleotide precursor oroticacid, has antiproliferative effects on human cells, but is especiallyuseful for treating infections with malarial parasites, e.g., Plasmodiumyoelii or Plasmodium falciparum, which are dependent on de novopyrimidine biosynthesis. Administration of uridine to mice treated with5-fluoroorotate attenuated host toxicity due to the latter withoutimpairing its antimalarial activity (Z M Gomez and P K Rathod,Antimicrob. Agents Chemother. 34:1371-1375 ('1490).

Dideoxycytidine (ddC) is also useful against retroviral infectionsincluding HIV; side effects of ddC include peripheral neuropathy, mouthulcers, and reduced platelet counts. The toxicity of ddC on humanmyeloid progenitor cells in culture can be ameliorated by deoxycytidinewithout thereby impairing the antiretroviral efficacy of ddC (K. Bhallaet al., AIDS 4:427-31 [1990]).

The methods disclosed in the prior art cited above for administeringthese pyrimidine nucleosides to modify chemotherapy in the clinicalsetting are neither practical (prolonged infusion of deoxycytidine oruridine via a central venous catheter requires hospitalization, risk ofinfection, and discomfort to the patient) or satisfactory (orallyadministered uridine is poorly absorbed; therapeutically adequate dosesof oral uridine produce diarrhea).

Commonly owned U.S. Pat. No. 438,493 demonstrates the use of acylatedderivatives of cytidine and uridine to increase blood cytidine oruridine levels.

Some acyl derivatives of pyrimidine nucleosides have been synthesizedfor use as protected intermediates in the synthesis of oligonucleotidesor nucleoside analogs, e.g. 5′-O-benzoyluridine, triacetylcytidine, andtriacetyluridine. See Sigma Chemical Company 1991 catalog, pages 155,980, and 981 respectively.

OBJECTS OF THE INVENTION

It is a primary object of this invention to provide a method foreffectively preventing or treating toxic symptoms of antiviral oranticancer chemotherapy, including but not limited to damage to thehematopoietic system and to gastrointestinal mucosa.

A further object of the invention is to provide compounds and methods topermit administration of higher doses of the chemotherapy agents.

A further object of the invention is to provide methods of increasingblood and tissue levels of uridine and cytidine, and their correspondingdeoxyribonucleosides deoxycytidine and deoxyuridine through oraladministration of a compound or compounds.

A further object of the invention is to provide a method for preventingor ameliorating gastrointestinal epithelium damage due to cytotoxicchemotherapy agents.

SUMMARY OF THE INVENTION

These and other objects of the invention are achieved by oral orparenteral administration of acylated derivatives of non-methylatedpyrimidine nucleosides, e.g. acylated derivatives of uridine,deoxyuridine, cytidine, or deoxycytidine, which are administered toanimals, including mammals such as humans. The administration of thesecompounds alone, or in combination, is useful in preventing orameliorating toxic effects of cytoreductive chemotherapy in animals.

Thus, the compounds of the invention, alone or in combinations, areuseful in the treatment of disorders of hematopoiesis induced bychemical agents; are useful as adjuncts to cancer and antiviralchemotherapy; and are useful for the treatment of other pathologicalconditions.

An important aspect of this invention is the discovery that acylderivatives of non-methylated pyrimidine nucleosides have unexpectedtherapeutic properties.

COMPOUNDS OF THE INVENTION

In all cases except where indicated, letters and letters with subscriptssymbolizing variable substituents in the chemical structures of thecompounds of the invention are applicable'only to the structureimmediately preceding the description of the symbol.

The compounds useful in attenuating toxicity due to anticancer orantiviral agents have the following general structures:

-   -   (1) An acyl derivative of uridine having the formula:

wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, provided that at least oneof said R substituents is not hydrogen, or a pharmaceutically acceptablesalt thereof.

-   -   (2) An acyl derivative of cytidine having the, formula:

wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, provided that at least oneof said R substituents is not hydrogen, or a pharmaceutically acceptablesalt thereof.

-   -   (3) An acyl derivative of deoxycytidine having the formula:

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.

-   -   (4) An acyl derivative of deoxyuridine having the formula:

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.

Compounds of the invention useful in ameliorating toxicity due toanticancer or antiviral chemotherapy agents include the following:

-   -   (5) An acyl derivative of uridine having the formula:

wherein R₁, R₂ and R₃ are the same, or different, and each is hydrogenor an acyl radical of

a. an unbranched fatty acid with 5 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine and ornithine,

c. a dicarboxylic acid having 3-22 carbon atoms,

d. a carboxylic acid selected from one or more of the group consistingof glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoicacid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid,betahydroxybutyric acid, orotic acid, and creatine.

-   -   (6) An acyl derivatives of cytidine having the formula:

wherein R₁, R₂, R₃, and R₄ are the same, or different, and each ishydrogen or an acyl radical of

a. an unbranched fatty acid with 5 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of phenylalanine, alanine, valine, leucine, isoleucine, tyrosine,proline, hydroxyproline, serine, threonine, cystine, cysteine, asparticacid, glutamic acid, arginine, lysine, histidine carnitine andornithine,

c. a dicarboxylic acid having 3-22 carbon atoms,

d. a carboxylic acid selected from one or more of the group consistingof glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoicacid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid,betahydroxybutyric acid, orotic acid, and creatine.

-   -   (7) An acyl derivative of deoxycytidine, having the formula

wherein R₁, R₂, and R₃ are the same, or different, and each is hydrogenor an acyl radical derived from

a. an unbranched fatty acid with 3 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine and ornithine,

c. nicotinic acid

d. a dicarboxylic acid having 3-22 carbon atoms, provided that not allof R₁, R₂, and R₃ are H, and where R₃ is not H, then R₁ and/or R₂ mayalso be acetyl, or a pharmaceutically acceptable salt thereof.

-   -   (8) An acyl derivative of deoxyuridine, having the formula

wherein R₁, R₂, and R₃ are the same, or different, and each is hydrogenor an acyl radical derived from

a. an unbranched fatty acid with 3 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine and ornithine,

c. nicotinic acid

d. a dicarboxylic acid having 3-22 carbon atoms, provided that not allof R₁, R₂, and R₃ are H, and where R₃ is not H, then R₁ and/or R₂ mayalso be acetyl, or a pharmaceutically acceptable salt thereof.

-   -   (9) An acyl derivative of uridine having the formula:

wherein at least one of R₁, R₂, or R₃ is a hydrocarbyloxycarbonyl moietycontaining 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

-   -   (10) An acyl derivative of cytidine having the formula:

wherein at least one of R₁, R₂, R₃ or R₄ is a hydrocarbyloxycarbonylmoiety containing 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

-   -   (11) An acyl derivative of deoxycytidine having the formula:

wherein at least one of R₁, R₂, or R₃ is a hydrocarbyloxycarbonyl moietycontaining 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

-   -   (12) An acyl derivative of deoxyuridine having the formula:

wherein at least one of R₁ or R₂ is a hydrocarbyloxycarbonyl moietycontaining 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

The invention, as well as other objects, features and advantages thereofwill be understood more clearly and fully from the following detaileddescription, when read with reference to the accompanying results of theexperiments discussed in the examples below.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to the use of acylated derivatives ofnon-methylated pyrimidine nucleosides, i.e. acylated derivatives ofuridine, deoxyuridine, cytidine, or deoxycytidine, such astriacetyluridine (TAU), to attenuate toxicity of chemotherapeutic agentsand antiviral agents in vivo. The invention also relates to theadministration of these pyrimidine nucleoside compounds, alone or incombinations, with or without other agents, to animals.

In the case of many antineoplastic and antiviral chemotherapy agents,exposure of affected cells to appropriate natural nucleosides canprevent or ameliorate damage to those cells. The compounds and methodsof the subject invention make it possible to reduce toxicity whilemaintaining therapeutic efficacy of the antiviral or antineoplasticagent, and conversely, to increase the dose of the chemotherapeuticagent while maintaining an acceptable degree of toxicity.

The present invention provides compounds and methods for treating orpreventing toxic symptoms of antiviral or anticancer chemotherapythrough oral or parenteral administration of acyl derivatives ofnon-methylated pyrimidine nucleosides.

A. DEFINITIONS

The term “non-methylated pyrimidine nucleoside” as used herein meansnaturally occurring nucleosides other than thymidine(5-methyldeoxyuridine) or 5-methylcytidine and other similarnaturally-occurring methylated nucleosides. Examples of non-methylatedpyridimidine nucleosides include uridine, cytidine, deoxyuridine, anddeoxycytidine.

The term “acyl derivative” as used herein means a derivative of anon-methylated pyrimidine nucleoside in which a substantially nontoxicorganic acyl substituent derived from a carboxylic acid is attached toone or more of the free hydroxyl groups of the ribose moiety of anon-methylated pyrimidine nucleoside with an ester linkage and/or wheresuch a substituent is attached to the amine substituent on thepyrimidine ring of cytidine or deoxycytidine, with an amide linkage.Such acyl substituents are derived from carboxylic acids which include,but are not limited to, compounds selected from the group consisting ofa fatty acid, an amino acid, nicotinic acid, dicarboxylic acids, lacticacid, p-aminobenzoic acid and orotic acid. Advantageous acylsubstituents are carboxylic acids which are normally present in thebody, either as dietary constituents or as intermediary metabolites.

The term “analog” as used herein means a nucleoside chemically modifiedin either the pyrimidine ring or the ribose (or deoxyribose) moiety by ameans other than acylation or attachment of other biologically labilesubstituents (e.g. phosphorylation of hydroxyl groups on the sugar).Specifically, nucleoside analogs, in the context of this invention, aredrugs with structural similarities to the naturally occurringnucleosides, but with antiviral, antineoplastic, or cytotoxicproperties. Examples of antineoplastic nucleoside analogs include butare not limited to the following: 5-fluorouracil (5-FU), 5-FU prodrugs(e.g. ftorafur, 5′-deoxyfluorouridine, carmofur), fluorouridine,2′-deoxyfluorouridine, prodrug derivatives of fluorouridine or2′-deoxyfluorouridine, fluorocytosine, arabinosyl cytosine, prodrugs ofarabinosyl cytosine, cyclocytidine, 5-aza-2′-deoxycytidine, arabinosyl5-azacytosine, 6-azauridine, azaribine, 6-azacytidine,trifluoro-methyl-2′-deoxyuridine, thymidine, and 3-deazauridine.Examples of antiviral nucleoside analogs include but are not limited tothe following: 5-ethyl-2′-deoxyuridine, 5-iodo-2′-deoxyuridine,5-bromo-2′-deoxyuridine, 5-methylamino-2′-deoxyuridine,arabinosyluracil, dideoxyuridine, dideoxycytidine,2′,3′-dideoxycytidin-2′-ene, 3′-deoxythymidin-2′-ene,3′-azido-2′,3′-dideoxyuridine, and 3′-azidodeoxythymidine (AZT). Analogsof pyrimidine nucleoside precursors, e.g. N-phosphonoacetyl-L-asparticacid (PALA), are encompassed by this term.

Some nucleoside analogs are considered to have structural similaritiesto particular naturally-occurring nucleosides. In the context of thecompounds of the invention, nucleoside analogs are divided into cytidineanalogs if they have an exocyclic amino group in the 4 position of thepyrimidine ring (an amino group in that position signifies thedistinction between cytidine and uridine). Nucleoside analogs that arespecifically analogs of cytidine include but are not limited to:fluorocytosine, arabinosyl cytosine, prodrugs of arabinosyl cytosine,cyclocytidine, 5-aza-2′-deoxycytidine, arabinosyl 5-azacytosine,6-azacytidine, and dideoxycytidine. Nucleoside analogs that arespecifically considered to be analogs of uridine include but are notlimited to: 5-fluorouracil (5-FU), 5-FU prodrugs (e.g. ftorafur,5′-deoxyfluorouridine, carmofur), fluorouridine, 2′-deoxyfluorouridine,prodrug derivatives of fluorouridine, prodrug derivatives of2′-deoxyfluorouridine, trifluoromethyl-2′-deoxyuridine, 6-azauridine,azaribine, 3-deazauridine, 5-ethyl-2′-deoxyuridine,5-iodo-2′-deoxyuridine, 5-bromo-2′-deoxyuridine,5-methylamino-2′-deoxyuridine, arabinosyluracil, and dideoxyuridine.Some cytotoxic nucleoside analogs are also specifically analogs ofthymidine, e.g. AZT.

The term “pharmaceutically acceptable salts” as used herein means saltswith pharmaceutically acceptable acid addition salts of the derivatives,which include, but are not limited to, sulfuric, hydrochloric, orphosphoric acids.

The term “coadministered” as used herein means that at least two of thecompounds of the invention are administered during a time frame whereinthe respective periods of pharmacological activity overlap.

The term “hydrocarbylcarbonyl” as used herein means an acyl radical of acarboxylic acid in which the atom adjacent to the carbonyl carbon atomis another carbon atom. The parent carboxylic acid may, for example, bea fatty acid, an aromatic acid (e.g. benzoate, nicotinoate, or theircongeners), an amino acid, a cycloalkylcarboxylic acid, or adicarboxylic acid.

The term “hydrocarbyloxycarbonyl” as used herein means an acyl radicalof a carboxylic acid in which the atom adjacent to the carbonyl carbonatom is oxygen which is furthermore covalently linked to another carbonatom. This can also be described as a radical of a carbonate ester of analcohol, which, when cleaved from a non-methylated pyrimidine nucleosidefollowing administration, degrades further into carbon dioxide and analcohol. Advantageous alcohols are those which are of low toxicity,particularly those which enter readily into normal metabolic oreliminative pathways.

The term “fatty acids” as used herein means aliphatic carboxylic acidshaving 2-22 carbon atoms. Such fatty acids may be saturated, partiallysaturated or polyunsaturated.

The term “amino acids” as used herein includes, but is not limited to,glycine, the L forms of alanine, valine, leucine, isoleucine,phenylalanine, tyrosine, proline, hydroxyproline, serine, threonine,cysteine, cystine, methionine, tryptophan, aspartic acid, glutamic acid,arginine, lysine, histidine, ornithine, hydroxylysine, carnitine, andother naturally occurring amino acids.

The term “dicarboxylic acids” as used herein means fatty acids with asecond carboxylic acid substituent.

The term “therapeutically effective amount” as used herein refers tothat amount which provides therapeutic effects for a given condition andadministration regime.

B. COMPOUNDS OF THE INVENTION

In all cases except where indicated, letters and letters with subscriptssymbolizing variable substituents in the chemical structures of thecompounds of the invention are applicable only to the structureimmediately preceding the description of the symbol.

The compounds useful in attenuating toxicity due to anticancer orantiviral agents have the following general structures:

-   -   (1) An acyl derivative of uridine having the formula:

wherein R₁, R₂, R₃ and R₄ are the same or different and each is hydrogenor an acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.

-   -   (2) An acyl derivative of cytidine having the formula:

wherein R₁, R₂, R₃ and R₄ are the same or different and each is hydrogenor an acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.

-   -   (3) An acyl derivative of deoxycytidine having the formula:

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.

-   -   (4) An acyl derivative of deoxyuridine having the formula:

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.

Compounds of the invention useful in ameliorating toxicity due toanticancer or antiviral chemotherapy agents include the following:

-   -   (5) An acyl derivative of uridine having the formula:

wherein R₁, R₂, and R₃ are the same, or different, and each is hydrogenor an acyl radical of

a. an unbranched fatty acid with 5 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine and ornithine,

c. a dicarboxylic acid having 3-22 carbon atoms,

d. a carboxylic acid selected from one or more of the group consistingof glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoicacid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid,betahydroxybutyric acid, orotic acid, and creatine.

(6) An acyl derivatives of cytidine having the formula:

wherein R₁, R₂, R₃, and R₄ are the same, or different, and each ishydrogen or an acyl radical of

a. an unbranched fatty acid with 5 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of phenylalanine, alanine, valine, leucine, isoleucine, tyrosine,proline, hydroxyproline, serine, threonine, cystine, cysteine, asparticacid, glutamic acid, arginine, lysine, histidine carnitine andornithine,

c. a dicarboxylic acid having 3-22 carbon atoms,

d. a carboxylic acid selected from one or more of the group consistingof glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoicacid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid,betahydroxybutyric acid, orotic acid, and creatine.

-   -   (7) An acyl derivative of deoxycytidine, having the formula

wherein R₁, R₂, and R₃ are the same, or different, and each is hydrogenor an acyl radical derived from

a. an unbranched fatty acid with 3 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine and ornithine,

c. nicotinic acid

d. a dicarboxylic acid having 3-22 carbon atoms,

provided that not all of R₁, R₂, and R₃ are H, and where R₃ is not H,then R₁ and/or R₂ may also be acetyl, or a pharmaceutically acceptablesalt thereof.

-   -   (8) An acyl derivative of deoxyuridine, having the formula

wherein R₁, R₂, and R₃ are the same, or different, and each is hydrogenor an acyl radical derived from

a. an unbranched fatty acid with 3 to 22 carbon atoms,

b. an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine and ornithine,

c. nicotinic acid

d. a dicarboxylic acid having 3-22 carbon atoms,

provided that not all of R₁, R₂, and R₃ are H, and where R₃ is not H,then R₁ and/or R₂ may also be acetyl, or a pharmaceutically acceptablesalt thereof.

-   -   (9) An acyl derivative of uridine having the formula:

wherein at least one of R₁, R₂, or R₃ is a hydrocarbyloxycarbonyl moietycontaining 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

-   -   (10) An acyl derivative of cytidine having the formula:

wherein at least one of R₁, R₂, R₃ or R₄ is a hydrocarbyloxycarbonylmoiety containing 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

-   -   (11) An acyl derivative of deoxycytidine having the formula:

wherein at least one of R₁, R₂, or R₃ is a hydrocarbyloxycarbonyl moietycontaining 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

-   -   (12) An acyl derivative of deoxyuridine having the formula:

wherein at least one of R₁ or R₂ is a hydrocarbyloxycarbonyl moietycontaining 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

Also encompassed by the invention are the pharmaceutically acceptablesalts of the above-noted compounds.

Advantageous compounds of the invention are fatty acid esters of uridineand deoxycytidine, especially those with 4 or fewer carbon atoms in theacyl substituent. Particularly advantageous compounds are fatty acidesters of uridine or deoxycytidine with 2 or 3 carbon atoms in the acylsubstituent.

Other advantageous compounds of the invention are hydrocarbyloxycarbonylderivatives of uridine and deoxycytidine, particularly those with 3 to 6carbon atoms in the hydrocarbyloxycarbonyl moiety.

In one embodiment of the invention, prodrugs of the compounds of theinvention with enhanced water solubility are prepared by attachingphosphate to a free hydroxyl group on the aldose moiety of the acylatednon-methylated pyrimidine nucleoside.

C. COMPOSITIONS OF THE INVENTION

Compositions of the invention include one or more of the above-notedcompounds along with a pharmaceutically acceptable carrier.

In another embodiment, the compositions of the invention include inaddition to one or more compounds of the invention and at least one ofthe following agents which enhance hematopoiesis: oxypurine nucleosides,congeners of oxypurine nucleosides, and acyl derivatives of oxypurinenucleosides and their congeners, e.g. fatty acid esters of guanosine ordeoxyguanosine (see U.S. Pat. No. 653,882, filed, Feb. 8, 1991, herebyincorporated by reference), a nonionic surfactant, an interleukin suchas IL-1, -2, -3, -4, -5, -6, -7, -8 (advantageously IL-1,3, or 6), acolony-stimulating factor, for example granulocyte colony-stimulatingfactor (G-CSF), granulocyte/macrophage colony-stimulating factor(GM-CSF), stem cell factor (SCF), erythropoietin (EPO), glucan,polyinosine-polycytidine, or any other agent having beneficial effectson hematopoiesis.

Acyl derivatives of oxypurine nucleosides which enhance hematopoiesisand which are optionally administered in conjunction with the compoundsof the invention have the following general structure:

-   -   R_(A)=H or an acyl radical of a carboxylic acid with 2 to 30        carbon atoms, and    -   R_(B)=H or an acyl radical of a carboxylic acid with 2 to 30        carbon atoms, and    -   Z=H, OH, ═O, or NHR_(C) where R_(C)=H or an acyl radical of a        carboxylic acid with 2 to 30 carbon atoms, and    -   L=H or OR_(D), where R_(D)=H or an acyl radical of a carboxylic        acid with 2 to 30 carbon atoms, and    -   M=H or OR_(E), where R_(E)=H or an acyl radical of a carboxylic        acid with 2 to 30 carbon atoms, with the proviso that at least        one of L and M is H, and    -   Q=H, a halogen, NHR_(F) where R_(F) is H or an acyl or alkyl        radical containing 1 to 10 carbon atoms, S divalently bound to        the carbon in which case the adjacent carbon-nitrogen double        bond is a single bond and an H is then attached to that        nitrogen, SR_(G) where R_(G) is H or an acyl or alkyl radical        containing 1 to 10 carbon atoms, O divalently bound to the        carbon, in which case the adjacent carbon-nitrogen double bond        is a single bond and an H is then attached to that nitrogen, or        OR_(H) where R_(H) is H or an acyl or alkyl radical containing 1        to 10 carbon atoms, and    -   the C—C bond between the 2′ and 3′ positions of the aldose        moiety is optionally present.

In another embodiment of the invention, an acylated non-methylatedpyrimidine nucleoside is formulated with a compound capable of enhancingthe uptake and phosphorylation of nucleosides into cells such as insulinor an insulinogenic carbohydrate.

In another embodiment of the invention, the composition comprises atleast one compound of the invention and an antiviral or antineoplasticagent (see detailed discussion of these agents in the section belowentitled Therapeutic Uses of the Compounds and Compositions of theInvention).

In another embodiment, the compositions of the invention comprise anacyl derivative of uridine or deoxyuridine and a compound capable ofinhibiting uridine phosphorylase. Uridine phosphorylase is the primaryenzyme involved in the catabolism of uridine, forming uracil and ribosephosphate. Administration of a compound which inhibits uridinephosphorylase will modify the pharmacokinetics and biological activityof uridine or deoxyuridine produced by deacylation of acylatedderivatives of these two non-methylated pyrimidine nucleosides. Examplesof suitable inhibitors of uridine phosphorylase include but are notlimited to 5-benzyl barbiturate or 5-benzylidene barbiturate derivativesincluding 5-benzyl barbiturate, 5-benzyloxybenzyl barbiturate,5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate,5-benzyloxybenzylacetyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate, and5-methoxybenzylacetylacyclobarbiturate, 2,2′-anhydro-5-ethyluridine, andacyclouridine compounds, particularly 5-benzyl substituted acyclouridinecongeners including but not limited to benzylacyclouridine,benzyloxy-benzylacyclouridine, aminomethyl-benzylacyclouridine,aminomethyl-benzyloxybenzylacyclouridine,hydroxymethyl-benzylacyclouridine, andhydroxymethyl-benzyloxybenzyl-acyclouridine. See also WO 89/09603 and WO91/16315, hereby incorporated by reference.

In another embodiment of the invention, the composition comprises anacyl derivative of a non-methylated pyrimidine nucleoside and a compoundwhich inhibits cellular uptake or excretion of non-methylated pyrimidinenucleosides, and thereby promotes maintenance of blood nucleoside levelsafter enzymatic deacylation of administered doses of acylatedderivatives of non-methylated pyrimidine nucleosides. Such modulators ofuridine transport or excretion include but are not limited todipyridamole, probenicid, lidoflazine or nitrobenzylthioinosine.

In another embodiment of the invention, the composition comprises anacyl derivative of cytidine and a compound capable of inhibiting theenzyme uridine phosphorylase. Inhibition of this enzyme is useful inconjunction with cytidine since cytidine is in part deaminated in thebloodstream after deacylation of its acyl derivatives, providing uridineto the tissues.

In another embodiment of the invention, the composition comprises anacyl derivative of cytidine or deoxycytidine and a compound capable ofinhibiting deoxycytidine deaminase. By inhibiting the deamination ofdeoxycytidine or cytidine, inhibitors of cytidine deaminase ordeoxycytidine deaminase such as tetrahydrouridine ortetrahydro-2′-deoxyuridine modify the efficacy of acyl derivatives ofcytidine or deoxycytidine. In another embodiment of the invention, aninhibitor of cytidine deaminase or deoxycytidine deaminase is used tomodify the toxicity of an antiviral or anticancer nucleoside analog (seeExample 11).

In another embodiment of the invention, especially for prevention ortreatment of damage to the gastrointesinal mucosa, the compositioncomprises an acyl derivative of a non-methylated pyrimidine nucleosideand an agent or agents with utility in promoting mucosal healing or inreducing discomfort. Examples of such agents include but are not limitedto sucralfate, mixtures of two or more deoxyribonucleosides as disclosedin U.S. Pat. No. 341,925, filed Apr. 21, 1989 (hereby incorporated byreference), allopurinol, antibiotics like chlorhexidine gluconate orlocal anesthetics like benzocaine.

In another embodiment of the invention, the composition comprises acombination of an acyl derivative of a non-methylated pyrimidinenucleoside and an orally-active antineoplastic nucleoside analog. Anadvantageous combination is an acyl derivative of uridine with an orallyactive fluorinated pyrimidines, especially prodrugs of 5-fluorouracil.In such compositions, the acyl derivative of a non-methylated pyrimidinenucleoside is mixed with (or otherwise adminstered with) theantineoplastic nucleoside analog in molar ratios, ranging from 1:1 to12:1. Molar ratios ranging from 2:1 to 8:1 are generally advantageous.Suitable orally-active fluorinated pyrimidines include Tegafur,5′-deoxyfluorouridine, 5-fluorouracil, 5-fluorouridine,2′-deoxy-5-fluorouridine,N⁴-trimethoxybenzoyl-5′-deoxy-5-fluorocytidine, or acyl derivativesthereof.

The compositions, depending on the intended use, are manufactured in theform of a liquid, a suspension, a tablet, a capsule, a dragee, aninjectable solution, a topical solution, or a suppository (seediscussion of formulation below).

As an alternative to formulation of compositions containing a compoundof the invention and another active agent (as discussed above), inanother embodiment, the compounds of the invention are coadministeredwith the other active agents.

D. THERAPEUTIC USES OF THE COMPOUNDS AND COMPOSITIONS OF THE INVENTION

The compounds of the invention are useful to prevent or treat damage tothe process of hematopoiesis and immune system function in animals. Thecompounds reduce damage to the process of hematopoiesis by minimizingloss in blood cell counts after bone marrow damage or suppression causedby antiviral or antineoplastic agents which affect nucleotidebiosynthesis, metabolism, or utilization. The compounds of the inventionare useful in treating humans; however, the invention is not intended tobe so limited, it being within the contemplation of the invention totreat all animals that experience a beneficial effect from theadministration of the active compounds of the invention.

The invention is furthermore embodied in the administration of apharmaceutical compound or composition of the invention, or incombinations, for the purpose of preventing, attenuating, orameliorating toxicity associated with administration of antiviral orantineoplastic agents which affect nucleotide biosynthesis, metabolism,or utilization.

Specific conditions where advantages are achieved using the compounds,compositions, and methods of the invention include situations where thehematopoietic system has suffered or is likely to suffer damage fromchemotherapy, particularly chemotherapy that affects nucleotidebiosynthesis, metabolism, or utilization. Such conditions includetreating animals, e.g. human patients, subjected to cytoreductive cancerchemotherapy or antiviral chemotherapy. Specifically included areveterinary applications requiring maintenance of blood cell counts.

The compounds and compositions are also useful for preventing ortreating damage caused by anticancer or antiviral chemotherapy agents toother tissues, including but not limited to gastrointestinal epithelium.For this purpose, the compounds and compositions are optionallyadministered orally, as a suppository, or parenterally.

By attenuating damage to the hematopoietic and immune systems caused byanticancer or antiviral chemotherapy, the compounds and methods of theinvention reduce the risk of susceptibility to opportunistic orsecondary infections (bacterial, viral, or fungal).

The efficacy of the compounds of the invention is enhanced bycoadministration of agents which stimulate the uptake andphosphorylation of pyrimidine nucleosides by cells. Such agents includehematopoietic growth factors (e.g. G-CSF, GM-CSF, SCF, acylatedoxypurine nucleosides and their congeners, erythropoietin, andinterleukins), insulin, and insulinogenic carbohydrates such as glucoseor glucose polymers.

Treatment of Complications Associated with Cancer Chemotherapy

The white blood cell counts, and particularly the neutrophil counts, ofpatients treated with standard anti-neoplastic chemotherapy agents(e.g., 5-fluorouracil, fluorodeoxyuridine, vinca alkaloids,cyclophosphamide and other nitrogen mustard alkylating agents,daunorubicin, doxorubicin, methotrexate, cytosine arabinoside,6-mercaptopurine, thioguanosine, podophyllotoxins, cisplatin orcombinations of such cytoreductive agents) are often greatly diminished.In the case of cytotoxic agents which act by affecting nucleotidebiosynthesis, metabolism, or utilization, daily administration (oral orparenteral) of an effective dose, (for example, 0.1-10.0 grams) of acompound of the invention such as triacetyluridine (or other acylderivatives of uridine, cytidine, deoxycytidine, or deoxyuridine) forseveral days reduces the severity of the neutropenia which typicallyoccurs several days to several weeks after chemotherapy is initiated.This reduces the likelihood of infection throughout the course oftreatment, and makes it possible for the patient to receive larger dosesof the chemotherapeutic agents and/or to receive repeated doses soonerthan comparable patients not treated with the uridine derivative(s).Similarly, chemotherapy-induced alterations in counts of other bloodcell types (lymphocytes, platelets, erythrocytes, etc.) are amelioratedby administration of the compounds and compositions of the invention.

Antineoplastic agents with which the compounds and methods of theinvention are particularly useful include: 5-fluorouracil (5-FU), 5-FUprodrugs (e.g. ftorafur, 5′-deoxyfluorouridine, carmofur),fluorouridine, 2′-deoxyfluorouridine, prodrug derivatives offluorouridine or 2′-deoxyfluorouridine, fluorocytosine (which also hasantifungal activity), arabinosyl cytosine, prodrugs of arabinosylcytosine, cyclocytidine, 5-aza-2′-deoxycytidine, arabinosyl5-azacytosine, N-phosphonoacetyl-L-aspartic acid (PALA), pyrazofurin,6-azauridine, azaribine, 6-azacytidine,trifluoro-methyl-2′-deoxyuridine, thymidine, and 3-deazauridine. Suchantineoplastic agents and various other therapeutic nucleoside analogsact by affecting nucleoside or nucleotide biosynthesis, utilization, ormetabolism; hence, amelioration of their toxic effects is accomplishedby administration of the pyrimidine compounds of the invention.

The compounds of the invention are administered before, during, and/orafter administration of the anti-neoplastic or antiviral agents.Typically, the compounds of the invention are administered after a doseof a cancer chemotherapy agent, as a means of “rescuing” normal tissuesafter administration of an effective antineoplastic dose of the agent.

Gastrointestinal epithelium is sensitive to cancer chemotherapy agentslike fluorouracil. Mucositis, stomatitis, or ulceration of thegastrointestinal mucosa are common side effects of cancer chemotherapy,resulting in discomfort, diarrhea, electrolyte imbalances and weightloss. The compounds of and compositions of the invention are useful inpreventing or treating damage to the gastrointestinal tract (includingthe mouth) caused by cancer chemotherapy agents. The compounds andcompositions of the invention are optionally administered for thispurpose as a solution or suspension in liquid form (as a mouthwash, as acomposition to be swallowed, or as an enema), as a capsule, dragee, ortablet, as an injectable solution, or as a suppository. Systemicadministration of the compounds and compositions of the invention alsoreduces damage to gastrointestinal mucosa caused by anticancer orantiviral nucleoside analogs.

Topical application of the compounds (e.g. to the scalp) of theinvention is useful for preventing chemotherapy-induced alopecia.

Acyl derivatives of uridine are advantageous in preventing or treatingtoxicity due to fluorouracil or related fluorinated analogs of uridine(e.g. fluorouridine or prodrugs thereof, fluorodeoxyuridine or prodrugsthereof, ftorafur, 5′-deoxyfluorouridine). For oral administration,advantageous acyl derivatives of uridine are those substituted withshort-chain fatty acids, (especially acetate) or with short chaincarbyloxycarbonates (e.g. ethoxycarbonate). Acyl derivatives of cytidineor deoxyuridine are also useful in treating toxicity due to fluorouracilor related fluorinated pyrimidine analogs.

In a typical therapeutic situation, a patient receives a dose offluorouracil, either as a single treatment agent or as part of a regimenalso involving administration of other antineoplastic drugs likemethotrexate, leucovorin, PALA, or cyclophosphamide. Several hours toone day after administration of the 5-FU, the patient receives an oraldose of 1 to 10 grams of triacetyluridine. The patient receivesadditional doses of 5-FU of similar size every 6 to 8 hours over thecourse of the next 2 to 4 days. The patient may receive additionalcourses of 5-FU plus TAU on a weekly basis or less frequently.

Acyl derivatives of uridine and, secondarily, cytidine, are alsoadvantageous for treatment or prevention of toxicity due toN-phosphonoacetyl-L-aspartic acid (PALA), pyrazofurin, 6-azauridine,azaribine, trifluoro-methyl-2′-deoxyuridine, and 3-deazauridine.

For modulating toxicity and efficacy of orally-active antineoplasticdrugs, particularly orally-active fluorinated pyrimidine or prodrugs offluorinated pyrimidines (such as 5′ deoxy-fluorouridine derivatives likeTegafur (5-fluoro-1-(tetrahydro-2-furfuryl)uracil),5′-deoxyfluorouridine, or related derivatives), acyl derivatives ofnon-methylated pyrimidine nucleosides may be used in several ways. Inone embodiment of the invention, the acyl derivative of a non-methylatedpyrimidine nucleoside is administered several hours to one day after adose of a fluorouracil prodrug such as Tegafur, similar to the situationwith parenteral administration of fluorouracil described above. In thiscontext, the delayed administration of the acyl derivative of anon-methylated pyrimidine nucleoside results in reduced toxicity of thefluorinated pyrimidine toward normal tissues. In another embodiment ofthe invention, the acyl derivative of a non-methylated pyrimidinenucleoside is administered at the same time as, or within about an hourof, the orally-active antineoplastic agent.

Tegafur, an orally active 5-fluorouracil prodrug, is currentlyadministered clinically in a formulation containing uracil in a molarratio of four parts uracil to one part tegafur. In this context, uracilpotentiates the antitumor efficacy of 5-fluorouracil produced bydegradation of tegafur (during and after absorption from the intestinaltract into the bloodstream) by competing with 5-fluorouracil for theenzyme which breaks down both pyrimidine molecules, dihydropyrimidinedehydrogenase. However, uracil also potentiates the toxicity of5-fluorouracil toward normal tissues, particularly the intestine.Gastrointestinal damage is the primary dose-limiting toxicity of amixture of tegafur and uracil. Co-administration of Tegafur (or otherorally active antineoplastic pyrimidine analogs) with an acyl derivativeof a non-methylated pyrimidine nucleoside results in the desirablepotentiation of the syste is toxicity of, for example, 5-fluorouracilderived from Tegafur, without potentiating its toxicity toward theintestinal mucosa as drastically as does uracil itself. The acylderivative of a non-methylated pyrimidine nucleoside and the orallyactive antineoplastic nucleoside analog are administered in ways and atdosages and molar ratios typically used for administration of uracil andan orally active antineoplastic agent. See, for example, U.S. Pat. No.4,328,229, hereby incorporated by reference. Higher doses of the orallyactive antineoplastic agent are also possible with the use of the acylderivatives of the subject invention. This embodiment of the inventionis experimentally demonstrated in Examples 13 and 14 below.

Acyl derivatives of deoxycytidine are advantageous in treating orpreventing toxicity due to antineoplastic nucleoside analogs that arespecifcally analogs of cytidine, e.g. arabinosyl cytosine or prodrugsthereof, cyclocytidine, 5-aza-2′-deoxycytidine, arabinosyl5-azacytosine, or 6-azacytidine. For oral administration, advantageousacyl derivatives of deoxycytidine are those substituted with short-chainfatty acids, especially acetate.

In a typical clinical situation involving the use of arabinosyl cytosineor related antineoplastic analogs of cytidine, which are primarilyutilized for treatment of leukemias, the acyl derivative(s) ofdeoxycytidine are administered orally, in a dose of 0.5 to 10 grams,either before or after administration of a dose of Ara-C is completed,or concurrently with the dose of Ara-C. Further doses of the acylderivative of deoxycytidine are administered every six to eight hoursfor 1 to 4 days. Repetitions of this treatment regimen are initiatedonce per week or less frequently, depending on the clinical response.

It is intended that the antineoplastic agents be used for treating thetypes of tumors for which they are normally utilized, e.g. Ara-C and itsrelated cytidine analogs are effective in leukemias, fluorouracil andrelated fluorinated uridine analogs are useful in treating tumors of thecolon, stomach, pancreas, and head-and-neck. In one embodiment, theantineolastic agents are administered in their normal doses, in whichcase the compounds of the invention primarily reduce the severity oftoxic side effects. In another embodiment, the antineoplastic agents areadministered in doses higher than normal, in which case the compounds ofthe invention permit safer administration of such higher,therapeutically aggressive doses of the anticancer drugs. Furthermore,the increases in therapeutic index of anticancer agents resulting fromuse of the compounds and compositions of the invention permit the use ofparticular antineoplastic agents for treating tumors for which they arenot currently standard therapy.

Treatment of Complications Associated with Viral Infection

HIV-infected patients, especially those whose infection has progressedto “acquired immunodeficiency syndrome” (AIDS), suffer from a variety ofsymptoms and diseases which result from and, in some cases, furtherexacerbate a severely compromised immune system. Many of these patientsare given antiviral chemotherapeutic agents, such as AZT, which alsohave detrimental effects on the body's immune function and uponhematopoiesis, further lowering resistance to infections of all kinds.Administration of the compounds of the invention—orally, intravenously,or by parenteral injection—raises the low blood cell counts due toantiviral chemotherapy agents, particularly those that modify nucleotidesynthesis, metabolism, or utilization, such as AZT or dideoxycytidine.Because anemia and greater susceptibility to infections are dose- andrate-limiting factors in chemotherapeutic treatment of AIDS patients,treatment of the patients with these compounds reduces chemotherapeuticside effects (and thus improves the quality of life) and, ifappropriate, permits a more intensive chemotherapeutic regimen to beemployed. AZT and dideoxycytidine produce deleterious side effects intissues other than bone marrow, including muscle and the peripheralnervous system. The compounds and compositions of the invention are alsouseful for treating or preventing such side effects.

Various antiviral nucleoside analogs other than AZT and dideoxycytidineare used to treat viral infections, including but not limited to HIV,herpes, or hepatitis. Examples of such agents include5-ethyl-2′-deoxyuridine, 5-iodo-2′-deoxyuridine,5-bromo-2′-deoxyuridine, 5-methylamino-2′-deoxyuridine,2′,3′-dideoxycytidin-2′-ene, 3′-deoxythymidin-2′-ene,3′-azido-2′,3′-dideoxyuridine, arabinosyluracil, dideoxyuridine,2′,3′-dideoxy-3′-fluorothymidine and(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC); see also WO89/09603, hereby incorporated by reference. The compounds of theinvention are used to treat or prevent deleterious side effects of theseand related other antiviral nucleoside analogs.

In treatment or prevention of toxicity due to antiviral chemotherapy,the compounds and compositions are administered prior to, during and/orafter administration of the antiviral agents. Typical antiviralchemotherapy regimens, especially for chronic viral infections such asHIV infection, involve daily (often multiple daily) administration ofthe antiviral agent or agents. The compounds of the invention areadministered, several times daily, daily, or less frequently, dependingon the clinical effect observed. In all cases, the antiviral drugs aretypically administered in their normal regimens for the types of viralinfections for which they are clinically useful. Treatment of patientsreceiving antiviral nucleoside analogs is undertaken either to reduceside effects of a standard dose or to permit administration of doses ofantiviral agents higher than are normally tolerated or utilized.

For treatment of toxicity due to AZT, acyl derivatives of either or bothuridine, cytidine, or deoxycytidine are useful. Particularlyadvantageous are acyl derivatives of deoxycytidine. For oraladministration, acyl derivatives of deoxycytidine, uridine, and cytidinesubstituted with short chain fatty acids (particularly acetate) or withshort chain carbyloxycarbonates (e.g. ethoxycarbonate), areadvantageous.

In a typical clinical situation, a patient receives AZT two to fourtimes daily, and must generally do so indefinitely. Doses of 1 to 10grams of acyl derivatives of uridine, cytidine, or deoxycytidine (ormixtures of two or all three) are administered orally once per week upto about four times per day, depending on the clinical response.

For treatment or prevention of toxicity due to dideoxycytidine, acylderivatives of deoxycytidine are advantageous.

Treatment of Complications Associated with Malarial Infection

Malarial parasites, e.g. Plasmodium yoelii or Plasmodium falciparum, aredependent upon de novo synthesis pathways for pyrimidine nucleotidebiosynthesis; mammalian cells in general can utilize either de novopathways or “salvage” pathways, through which advanced nucleotideprecursors such as uridine or cytidine are incorporated intointracellular nucleotide pools.

5-Fluoroorotate, an analog of the pyrimidine nucleotide precursor oroticacid, is toxic toward malarial parasites which are dependent on de novopyrimidine biosynthesis. Other inhibitors of de novo pyrimidinebiosynthesis, such as PALA, pyrazofurin or 6-azauridine are alsosimilarly toxic toward malaria parasites. Inhibitors of pyrimidinebiosynthesis, including especially fluoroorotate, are also toxic towardmammals. However, administration of uridine to mammals treated with5-fluoroorotate (or other inhibitors of pyrimidine biosynthesis)attenuates host toxicity due to the latter without impairing itsantimalarial activity. Orally active agents which elevate blood uridinelevels are advantageous sources of uridine in this context. Such agentsinclude the acyl derivatives of uridine or cytidine of the invention. Intreatment of malaria, an effective anti-malarial dose of fluoroorotateis administered. Before, after, or at the same time as fluoroorotateadministration, an acyl derivative of uridine or cytidine(triacetyluridine is particularly advantageous) is administered, in adose sufficient to attenuate fluoroorotate toxicity. Typical doses of anacylated uridine or cytidine derivative such as triacetyl uridine rangefrom 1 to 10 grams, administered as often as needed to minimizedfluoroorotate toxicity, e.g. one to four times per day. Doses offluoroorotate or uridine are optionally repeated as necessary toovercome the malarial infection and to reduce host toxicityrespectively.

E. ADMINISTRATION AND FORMULATION OF COMPOUNDS AND COMPOSITIONS OF THEINVENTION

The compounds and compositions of the invention are administered orally,by parenteral injection, intravenously, topically, or by other means,depending on the condition being treated.

The optimal doses and dose schedules for triacetyluridine (or other acylderivatives of uridine, cytidine, deoxycytidine or deoxyuridine) arereadily determined by one skilled in the art, by monitoring thetherapeutic effect.

The compounds and compositions of the invention are administeredchronically or intermittently. The compounds and compositions areadministered prior to, during, or after an exposure to cytoreductive orantiviral chemotherapy agents, depending on the characteristics of thetoxicity of the chemotherapy agents.

Advantageous acyl derivatives of uridine, cytidine, deoxycytidine, ordeoxyuridine for oral administration are those substituted with shortchain (2-6 carbon) fatty acids on the hydroxyl groups of their ribose ordeoxyribose rings. Also advantageous for oral administration arepyrimidine nucleosides substituted on their hydroxyl groups withhydrocarbyloxycarbonyl radicals containing 3-7 carbon atoms.

Dosages for orally adminstered acyl derivatives of uridine, cytidine,deoxycytidine or deoxyuridine typically range from 0.5 to 20 grams perday, most commonly 2 to 10 grams per day.

Powdered acyl derivatives of uridine, cytidine, deoxycytidine ordeoxyuridine are administered orally in capsule or tablet form, althoughsolutions, emulsions, or suspensions are also useful for oraladministration.

The compounds of the invention are optionally formulated inbiodegradable, bioerodible, or other gradual-release matrices forsustained release of the compounds after oral administration orsubcutaneous implantation. In the case of intravenous or intramuscularinjection, the compounds are optionally formulated in liposomes.

The pharmacologically active compounds optionally are combined withsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds. Theseare administered as tablets, dragees, capsules, and suppositories. Thecompositions are administered, for example, orally, rectally, vaginally,or released through the buccal pouch of the mouth, and are optionallyapplied in solution form by injection, orally or by topicaladministration. The compositions may contain from about 0.1 to 99percent, preferably from about 50 to 90 percent, of the activecompound(s), together with the excipient(s).

For parenteral administration by injection or intravenous infusion, theactive compounds are suspended or dissolved in aqueous medium such assterile water or saline solution. Injectable solutions or suspensionsoptionally contain a surfactant agent such as polyoxyethylenesorbitanesters, sorbitan esters, polyoxyethylene ethers, or solubilizing agentslike propylene glycol or ethanol. The solution typically contains 0.01to 5% of the active compounds. The active compounds optionally aredissolved in pharmaceutical grade vegetable oil for intramuscularinjection. Such preparations contain about 1% to 50% of the activecompound(s) in oil.

Suitable excipients include fillers such as sugars, for example lactose,sucrose, mannitol or sorbitol, cellulose preparations and/or calciumphosphates, for example tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch or potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethyl cellulose and/or polyvinyl pyrrolidone.

Auxiliaries include flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions are used,which optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigmentsare optionally added to the tablets or dragee coatings, for example, foridentification or in order to characterize different compound doses.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral useare obtained by combining the active compound(s) with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Other pharmaceutical preparations which are useful for oral deliveryinclude push-fit capsules made of gelatin, as well as soft-sealedcapsules made of gelatin and a plasticizer such as glycerol or sorbitol.The push-fit capsules contain the active compound(s) in the form ofgranules which optionally are mixed with fillers such as lactose,binders such as starches and/or lubricants such as talc or magnesiumstearate, and, optionally stabilizers. In soft capsules, the activecompounds are preferably dissolved or suspended in suitable liquids suchas fatty oils, liquid paraffin, or polyethylene glycols. In addition,stabilizers optionally are added.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water soluble form, for example,water soluble salts. In addition, suspensions of the active compounds asappropriate in oily injection suspensions are administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortri-glycerides. Aqueous injection suspensions optionally includesubstances which increase the viscosity of the suspension which include,for example, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension optionally contains stabilizers.

In another embodiment, the active compounds are formulated as part of askin lotion for topical administration. Suitable lipophilic solvents orvehicles include fatty oils, for example sesame oil or coconut oil, orsynthetic fatty acid esters, for example ethyl oleate or triglycerides.

In another embodiment, the active compounds are formulated in vehiclessuitable for direct treatment of gastrointestinal mucosa. Examplesinclude mouthwashes, liquids (solutions or suspensions) to be swallowed,or viscous fluids (e.g. solutions of methylcellulose,carboxymethylcellulose, xanthan gum, etc.) which are administered orallyor rectally.

Other pharmaceutical preparations which are used rectally, especiallyfor treatment of the colon and rectum, include, for example,suppositories which consist of a combination of active compounds with asuppository base. Suitable suppository bases are, for example, naturalor synthetic triglycerides, paraffin hydrocarbons, polyethylene glycolsor higher alkanols. In addition, gelatin rectal capsules which consistof a combination of the active compounds with a base are useful. Basematerials include, for example, liquid triglycerides, polyethyleneglycols, or paraffin hydrocarbons.

F. SYNTHESIS OF THE COMPOUNDS OF THE INVENTION

Acylated derivatives of non-methylated pyrimidine nucleosides aresynthesized by reacting a pyrimidine nucleoside with an activatedcarboxylic acid. An activated carboxylic acid is one that has beentreated with appropriate reagents to render its carboxylate carbon moresusceptible to nucleophilic attack than is the case in the originalcarboxylic acid. Examples of useful activated carboxylic acids forsynthesis of the compounds of the invention are acid chlorides, acidanhydrides, n-hydroxysuccinimide esters, or carboxylic acids activatedwith BOP-DC. Carboxylic acids are alternatively linked to pyrimidinenucleosides with coupling reagents like dicyclohexylcarbodiimide (DCC).

During preparation of the acyl compounds of the invention, when the acidsource of the desired acyl derivative has groups which interfere withthe acylation reactions, e.g., hydroxyl or amino groups, these groupsare blocked with protecting groups, e.g., t-butyldimethylsilyl ethers ort-BOC groups, respectively, before preparation of the anhydride. Forexample, lactic acid is converted to 2-t-butyldimethyl-siloxypropionicacid with t-butyl-dimethylchlorosilane, followed by hydrolysis of theresulting silyl ester with aqueous base. The anhydride is formed byreacting the protected acid with DCC. With amino acids, the N-t-BOC orN-CBZ derivative is prepared, using standard techniques, which is thenconverted to the anhydride with DCC. With acids containing more than onecarboxylate group (e.g., succinic, fumaric, or adipic acid) the acidanhydride of the desired dicarboxylic acid is reacted with a pyrimidinenucleoside in pyridine or pyridine plus dimethylformamide ordimethylacetamide.

Amino acids are coupled to the exocyclic amino groups of cytosine anddeoxycytosine, and to hydroxyl groups on the aldose moiety of pyrimidinenucleosides, by standard methods using DCC in a suitable solvent,particularly a mixture of (i) methylene chloride and (ii)dimethylacetamide or dimethylformamide.

Carbyloxycarbonyl derivatives of non-methylated pyrimidine nucleosidesare prepared by reacting the nucleoside with the appropriatecarbylchloroformate in a solvent such as pyridine or pyridine plusdimethylformamide under anhydrous conditions. The solvent is removedunder vacuum, and the residue is purified by column chromatography.

It will be obvious to the person skilled in the art that other methodsof synthesis may be used to prepare the compounds of the invention.

The following examples are illustrative, but not limiting of the methodsand compositions of the present invention. Other suitable modificationsand adaptations of a variety of conditions and parameters normallyencountered in clinical therapy which are obvious to those skilled inthe art are within the spirit and scope of this invention.

EXAMPLES Example 1 Oral Administration of Triacetyluridine AmelioratesHematologic Toxicity of 5-Fluorouracil

Purpose

This study was undertaken in order to determine if oral administrationof TAU could rescue mice from 5-FU toxicity more effectively than oraladministration of uridine itself. Bone marrow cellularity and peripheralblood cell counts were used as an index of 5-FU toxicity.

Methods

Forty-five female Balb/C mice (20 grams each) were given 5-fluorouracil(150 mg/kg, i.p.) at 12:00 noon on the initial day of the experiment.These animals were then divided into 5 groups: control (water, p.o.),oral uridine at 400 mg/kg/dose, oral uridine at 800 mg/kg/dose,parenteral (i.p.) uridine at 400 mg/kg/dose, and oral TAU at 500mg/kg/dose.

Two hours after administration of 5-FU, the rescue treatments withuridine or triacetyluridine were begun. Groups received their designatedtreatment at 2:00 p.m., 4:00 p.m., and 6:00 p.m. on the day of 5-FUadministration, and at 9 a.m., 11:00 a.m., 1:00 p.m., 3:00 p.m. and 5:00p.m. on the following day. Each dose of uridine or TAU was administeredin 0.2 ml of water by gavage or in 0.2 ml of saline by i.p. injection,as appropriate.

Seven days after administration of 5-FU, blood (0.2-0.3 ml) from fivemice in each group was collected from the suborbital sinus into EDTA forsubsequent differential blood cell counting. Mice were sacrificed bycervical dislocation; femurs were removed, and their cell contents wereexpelled for counting; spleens were also removed and weighed. Thirteendays after administration of 5-FU, the remaining four mice in each groupwere bled, sacrificed, and their spleens removed.

Results

Day Seven

5-FU administration resulted in declines in counts of all blood celltypes examined. Seven days after administration of 5-FU, neutrophil,lymphocyte, and platelet counts in animals treated with oral TAU weresignificantly higher than in control animals (Table 1). It isparticularly noteworthy that leukocyte counts in the mice that receivedTAU were higher than in mice that received an equimolar dose of uridineby intraperitoneal injection. Cell counts in the mice that received oralTAU were also higher than in mice that received uridine orally at eitherequimolar (400 mg/kg/dose) or twice equimolar doses (800 mg/kg/dose).

Platelet counts were within the normal range (700-800 K/μl) in the micethat received oral TAU and i.p. uridine. They were subnormal in theother groups, and lowest in the control mice (Table 1).

Bone marrow cell counts were significantly greater in the mice treatedwith TAU orally and with uridine parenterally than any of the othergroups (Table 2)

Day Eleven

Importantly, neutrophil and RBC levels were higher in the group thatreceived TAU than in the other treatment groups, including mice thatreceived an equimolar dose of uridine by intraperitoneal injection(Table 3).

Spleen weight is an index of hematopoietic activity in mice recoveringfrom bone marrow damage. Eleven days after 5-FU, spleen weight wassignificantly higher in mice given oral TAU compared to the othertreatment groups; spleens were smallest in the control group (Table 4).

Conclusion

The results of this study show that oral administration of TAU rescuesmice from 5-FU toxicity more effectively than oral administration ofequimolar and twice equimolar uridine itself, and more effectively thanan equimolar dose of uridine given by intraperitoneal injection.

TABLE 1 Blood cell counts 7 days after 5-FU Lympho- WBC Neutrophilscytes Platelets RBC Control 3.4 ± .1  0.24 ± .05  3.18 ± .07  387 ± 72 8.05 ± .11 K/μl Urd 400 4.4 ± .5* 0.48 ± .23  3.01 ± .77  434 ± 114 7.66 ± .12 oral Urd 800 4.6 ± .4* 0.88 ± .22*  3.76 ± .27  646 ± 108 8.24 ± .26 oral Urd 400 4.6 ± .4* 0.83 ± .24*  3.80 ± .30  773 ± 54**8.06 ± .05 i.p. TAU 5.9 ± .4* 1.20 ± .23** 4.72 ± .36** 723 ± 57** 8.27± .16 500 oral All blood cell count units are K/μl except RBC's, whichare M/μl * = greater than control value, P < .05 ** = greater thancontrol value, P < .01

TABLE 2 Marrow cell counts and spleen weight 7 days after 5-FU Marrowcell counts Spleen weight Control 1.5 ± .2 × 10⁶/femur 71 ± 2.6 mg Urd400 oral 1.6 ± .2 × 10⁶   74 ± 2.4 Urd 800 oral 2.9 ± .9 × 10⁶   74 ±6.4 Urd 400 i.p. 5.0 ± .7 × 10⁶** 78 ± 5.5 TAU 500 oral 3.0 ± .5 × 10⁶* 78 ± 3.5 * = greater than control value, P < .05 ** = greater thancontrol value, P < .01

TABLE 3 Blood cell counts 11 days after 5-FU Lympho- WBC Neutrophilscytes Platelets RBC Control 5.2 ± .3 0.48 ± .12  4.68 ± .11 2280 ± 1937.31 ± .05  Urd 400 4.6 ± .5 0.84 ± .19  3.65 ± .27 1940 ± 177 7.44 ±.14  oral Urd 800 4.9 ± .4 0.82 ± .19  4.07 ± .38 2127 ± 143 7.33 ± .16 oral Urd 400 5.1 ± .5 1.20 ± .21*  3.84 ± .38 1706 ± 88  7.78 ± .22 i.p. TAU 5.4 ± .5 1.89 ± .12** 3.29 ± .36 1446 ± 160 8.05 ± .16** 500oral All blood cell count units are K/μl except RBC's, which are M/μl *= greater than control value, P < .05 ** = greater than control value, P< .01

TABLE 4 Spleen weight 11 days after 5-FU Spleen weight Control 76 ± 7 mgUrd 400 oral 103 ± 15 Urd 800 oral 99 ± 6* Urd 400 i.p. 104 ± 6* TAU 500oral 143 ± 11** * = greater than control value, P < .05 ** = greaterthan control value, P < .01

Example 2 TAU Accelerates Hematopoietic Recovery in 5-FU-Treated Animalsin a Dose Dependent Manner

Purpose

The purpose of this experiment was to confirm and extend the previousfindings that orally administered TAU accelerates hematopoietic recoveryin mice treated with 5-fluoruracil (5-FU), and to observe therelationship between increasing doses of TAU and the responses of thehematopoietic system of these 5-FU-treated mice.

Methods

Seventy female Balb/C mice weighing approximately twenty grams were eachgiven an i.p. injection of 5-FU (150 mg/kg) at 1:00 p.m. on the initialday of the study. These animals were then divided into five differenttreatment groups: control (water), and oral TAU at doses of 100, 250,500, and 1,000 mg/kg/treatment. The test compounds were then given at3:00, 5:00, 7:30, and 10:00 p.m. on the day of 5-FU administration; at9:00 a.m., 11:00 a.m., and 1:00, 3:00, 6:00, and 10:00 p.m. thefollowing day; and a final administration at 11:00 a.m. two daysfollowing the single 5-FU injection. Each treatment was given orally ina volume of 0.2 ml water (by gavage), except the highest dose of TAUwhich was administered in 0.4 ml of water.

On days seven and eleven following 5-FU administration, blood (0.2-0.3ml) was collected from seven mice in each group by retro-orbitalbleeding into EDTA for subsequent differential blood cell counting. Micewere sacrificed by cervical dislocation; their right femurs removed andthe contents expelled for cell counting; and their spleens removed andweighed.

Results

Day Seven.

With increasing doses of TAU there were increasing numbers of nucleatedcells in the bone marrow. While the number of such cells in the groupreceiving TAU 100 was approximately equal to that in the control group,differences in bone marrow cellularity were significantly greater in theTAU 500 and TAU 1,000 groups compared that of controls (Table 5).

Treatment with TAU at doses of 500 and 1,000 mg/kg/treatment resulted inwhite blood cell counts significantly greater than those in the controlgroup.

Total neutrophil counts also appeared to increase in a dose-dependentmanner, reaching approximately three-fold higher levels in the TAU 1,000group compared to controls.

In both the TAU 500 and TAU 1,000 groups lymphocyte counts weresignificantly elevated compared to those in the control group.

Platelet counts were significantly elevated in the TAU 250, 500, and1,000 groups. The dose-response curve appeared to plateau at about 500mg/kg/treatment (Table 6).

TABLE 5 Effect of increasing doses of TAU on hematopoiesis in mice sevendays after 5-FU administration. All blood cell counts are K/μl. MarrowWBC Neut Lym Control 1.70 ± .35  2.46 ± .13  .026 ± .011 2.43 ± .13  TAU100 1.77 ± .18  2.47 ± .04  .014 ± .008 2.46 ± .04  TAU 250 2.91 ± .48 2.76 ± .20  .027 ± .012 2.71 ± .20  TAU 500 3.93 ± .39* 4.06 ± .14* .037± .020 3.99 ± .15* TAU 1000 4.42 ± .34* 3.57 ± .25* .071 ± .027 3.44 ±.23* *indicates different from control, p < .01

TABLE 6 Effect of increasing doses of TAU on red blood cells (RBC) andplatelets (PLT) in mice seven days after 5-FU administration. RBC PLT(M/μl) (K/μl) Control 8.38 ± .18 308 ± 52  TAU 100 8.20 ± .07 440 ± 46 TAU 250 8.51 ± .06 601 ± 46** TAU 500 8.43 ± .16 712 ± 42** TAU 10008.65 ± .15 715 ± 35** **indicates different from control, p < .01

Day Eleven.

Spleen weight was somewhat elevated at every dose of TAU compared tocontrols, but reached statistical significance only in the TAU 250 group(104.2±3.5 mg versus 79.7±3.1; p<0.05)

Total neutrophil counts were significantly greater than control valuesin the TAU 250, 500, and 1,000 groups.

Red blood cell counts were significantly increased in the TAU 500 group(8.72±0.18×10⁶ per microliter) and the TAU 1000 group (8.63±0.16×10⁶ permicroliter) compared to controls (7.90±0.09×10⁶ per microliter). Thehematocrit followed a similar pattern (Table 7).

TABLE 7 Effect of increasing doses of TAU on hematocrit (HCT) in miceseven (7 d) and eleven (11 d) days after 5-FU administration. ControlT100 T250 T500 T1000 HCT 38.50 ± .88  37.77 ± .37 40.04 ± .38 39.50 ±.61 40.84 ± .68 (7 d) HCT 36.94 ± 0.51 36.80 ± .88 37.47 ± .83 41.16 ±.88 41.55 ± .77 (11 d) p < .01 p < .001Conclusions

The results of this experiment confirm and extend the previous findingsthat treatment of animals receiving the chemotherapeutic agent 5-FU withTAU dramatically reverses the detrimental effects of 5-FU on thehematopoietic system, and that it does so in a dose-dependent manner.

Example 3 Acyl Derivatives of Uridine Ameliorate Bone Marrow Toxicity of5-Fluorouracil

Purpose

The purpose of this experiment was to test and compare the efficacy ofuridine and derivatives of uridine in attenuating damage to thehematopoietic system of mice caused by the chemotherapeutic agent5-fluorouracil (5-FU).

Methods

Ninety-eight female Balb/C mice weighing approximately 20 grams eachwere given a one-time 150 mg/kg injection (i.p.) of 5-FU at 1 p.m. onthe initial day of the study. These animals were then divided into sevengroups: control (saline), uridine (300 mg/kg/treatment),triacetyluridine (TAU; 455 mg/kg/treatment), benzoyluridine (BU; 428mg/kg/treatment), ethoxycarbonyl (ECU; 389 mg/kg/treatment),octanoyluridine (OU; 455 mg/kg/treatment), and valeryluridine (VU; 403mg/kg/treatment). All of these doses are equimolar, and wereadministered in a volume of 0.4 ml by i.p. injection. The groups weretreated at 3:30, 6:00, and 8:30 p.m. on the initial day with theirrespective agents. On the following day these compounds wereadministered at 9:30 a.m., 12:00 noon, 2:30, and 5:00 p.m. One finaltreatment was given on the next day at 10:00 a.m.

On days seven and eleven following 5-FU administration, blood (0.2-0.3ml) was collected from seven mice in each group by retro-orbitalbleeding into EDTA for subsequent differential blood cell counting. Micewere sacrificed by cervical dislocation; their right femurs removed andthe contents expelled for cell counting; and their spleens removed andweighed.

Results

Day Seven.

Even at this first time point each of the uridine derivativesaccelerated one or more aspects of hematopoietic recovery following 5-FUdamage. Thus, spleen weight was elevated in all of the groups comparedto saline controls. These differences reached statistical significance(p<0.05) in the uridine (80.4±7.8 mg), ECU (75.7±5.0 mg), and VU(69.1±1.7 mg) groups when compared to controls (62.7±1.8).

TAU increased bone marrow cellularity by 40% over control values(4.50±0.77×10³ per microliter versus 2.78±0.45×10³ per microliter,respectively).

White blood cell counts were significantly elevated in both theECU-treated (7.26±0.31×10³ per microliter; p<0.01) and VU-treated(6.57±0.49×10³ per microliter; p<0.05). groups compared to salinecontrols (4.60±0.70×10³ per microliter).

Platelet counts were significantly greater in the groups treated withuridine (785.3±57.5×10³ per microliter; p<0.02), BU (829.6±×10³ permicroliter; p<0.01), and VU (825.7±26.7×10³ per microliter; p<0.002)than those in the saline-treated controls (523.2±71.4×10³ permicroliter).

There was a trend toward higher total neutrophil counts in nearly all ofthe treatment groups, but only VU (0.141±0.027×10³ per microliter) wasactually statistically significantly greater (p<0.002) than salinecontrols (0.013±0.009×10³ per microliter).

Lymphocyte counts were significantly greater in the groups treated withECU (7.19±0.32×10³ per microliter; p<0.01) and VU (6.42±0.49×10³ permicroliter) than in the saline-treated controls (4.59±0.70×10³ permicroliter).

At the doses used in this particular experiment, octanoyluridine, havingthe longest carbon chain of any of the other derivatives, proved to besomewhat detrimental. There were not enough animals from this group toprovide day 11 data. However, in dose optimization studies (see Example3A), octanoyluridine administered at a lower dose showed very beneficialeffects on hematopoietic recovery following 5-FU.

Day Eleven.

Virtually every index of hematopoietic function, including spleenweight, white blood cell counts, red blood cell counts, hematocrit,neutrophil counts, and lymphocyte count, was significantly improved bytreatment with the uridine derivatives used in this experiment (Table8). Spleen weights were elevated above those of controls (94.7±7.4) ineach treatment group, reaching statistical significance (p<0.05) in theuridine (121.6±9.7), BU (126.9±12.3), and VU (139.4±8.0) groups.

Conclusions

A wide variety of the uridine derivatives of the invention are effectivein ameliorating damage caused by administration of the chemotherapeuticagent 5-FU.

TABLE 8 Effect of uridine and derivatives of uridine on hematopoiesis inmice eleven days after 5-FU administration. White blood cell counts(WBC), neutrophil counts (Neut), and lymphocyte counts (lym) are allexpressed in K/μl; red blood cell counts (RBC) in M/μl. WRC RBC HCT %Neut Lym Con 3.37 ± .68  7.11 ± .1 32.97 ± 0.4 .09 ± .047  3.3 ± .7  Urd5.20 ± .47*  7.32 ± .2 34.67 ± 0.9 .60 ± .180** 4.6 ± .4  TAU 6.23 ±.55** 7.35 ± .2 35.01 ± 1.2 .55 ± .164** 5.6 ± .4* BU 6.20 ± .49**  7.62± .1**  36.87 ± 0.6** 1.04 ± .295**  5.1 ± .4* ECU 5.51 ± .64*   7.65 ±.2*  37.54 ± 1.0** .66 ± .166** 4.8 ± .5* VU 6.31 ± .42**  7.83 ± .1** 38.31 ± 0.8** .97 ± .136** 5.2 ± .4* *indicates different from Control,p < .05 **indicates different form Control, p < .01 Con = control; Urd =uridine; TAU = triacetyluridine; BU = benzoyluridine; ECU =ethoxycarbonyluridine; VU = valeryluridineCon=control; Urd=uridine; TAU=triacetyluridine;BU=benzoyluridine;ECU=ethoxycarbonyluridine; VU=valeryluridine

Example 3A Octanoyl Uridine Attenuates Hematological Toxicity of 5-FU

Purpose

The purpose of this experiment was to test the efficacy ofoctanoyluridine (Oct-U) in ameliorating the toxic effects of5-fluorouracil (5-FU) on hematopoiesis.

Methods

Fourteen Balb/C female mice weighing approximately 20 grams each weregiven a one-time 75 mg/kg i.p. injection of 5-FU at 11:00 a.m. on theinitial day of the study. Half of these animals were subsequentlytreated with Oct-U (100 mg/kg/treatment, i.p.), while the other half(controls) were injected with physiological saline. Administration ofOct-U and saline occurred at 2:30, 4:30, and 7:00 p.m. on the initialday, and at 9:30 a.m., 12:00 noon, 2:30, and 5:00 p.m the following day.An additional group of seven mice (basals) received no 5-FU and notreatments.

Results

Administration of 5-FU resulted in statistically significant damage tothe hematopoietic system as measured by each and every index employed inthis study, including spleen weight, bone marrow cellularity, WBC-count,total neutrophil count, lymphocyte counts, RBC count, hematocritpercent, and platelet count (Tables 9 and 10). Subsequent treatment ofmice receiving 5-FU with Oct-U resulted in substantial improvement ineach and all of these parameters of hematopoietic function (Table 9 and10).

Conclusion

Treatment of mice receiving 5-FU with octanoyl uridine ameliorates thetoxic effects of 5-FU on hematopoiesis.

TABLE 9 Effect of octanoyluridine on bone marrow cellularity andmyelopoiesis in mice seven days after 5-FU administration. All countsare in thousands per microliter. Marrow WBC Neut Lym Basal 8.96 ± .32 6.89 ± .25 1.85 ± .24  4.78 ± .23  Control 4.16 ± .40*  4.04 ± .19* 0.26 ± .03*  3.74 ± .19*  Oct-U 7.73 ± .68** 5.96 ± .47** 1.19 ± .22**4.62 ± .29** *indicates p < .01 versus basals **indicates p < .01 versuscontrols

TABLE 10 Effect of octanoyluridine on red blood cells (RBC), platelets(PLT), and spleen weight in mice seven days after 5-FU administration.RBC PLT Spleen weight (M/μl) (K/μ) (mg) Basal 8.56 ± .17 897 ± 21  83.6± 1.7  Control  7.98 ± .13* 963 ± 13*  75.4 ± 2.4*  Oct-U 8.13 ± .141243 ± 59** 87.2 ± 3.7** *indicates p < .05 versus basal **indicates p <.05 versus control

Example 4 Plasma Uridine Levels after Administration of Acyl Derivativesof Uridine

Methods

Plasma uridine levels were determined in mice at various times (15minutes, 30 minutes, 1 hour, and 2 hours) after administration of theacyl derivatives of uridine utilized in Example 3 for attenuation oftoxicity caused by 5-FU. Groups of mice (n=3 per compound per timepoint) received intraperitoneal injections of uridine (300 mg/kg),triacetyluridine (TAU; 455 mg/kg/treatment), benzoyluridine (BU; 428mg/kg/treatment), ethoxycarbonyluridine (ECU; 389 mg/kg/treatment),octanoyluridine (OU; 455 mg/kg/treatment), and valeryluridine (VU; 403mg/kg/treatment). The doses of the acyl derivatives of uridine are themolar equivalent of 300 mg/kg uridine. At the appropriate time points,blood samples (200 μl) were taken from mice via the retro-orbital sinusand immediately centrifuged. 75 μl of the resulting plasma wasdeproteinized with 2 volumes of methanol followed by centrifugation. Thesupernatant was lyophilized and reconstituted with 50 mM potassiumphosphate buffer, pH 6.0, and analyzed for uridine content by HPLC on areverse phase (C₁₈) column. Uridine was separated from other plasmacomponents in 50 mM potassium phosphate buffer, pH 6.0, with a methanolgradient (2% to 35% over 15 minutes). Uridine was detected andquantified by UV absorbance at 260 nM.

Results

Administration of all of the acyl derivatives of uridine tested resultedin increased plasma uridine levels, as shown in Table 11. Plasma uridinelevels in control animals (mice that received no exogenous uridine orcytidine derivatives) were 1.1±0.1 μM.

TABLE 11 Plasma uridine concentration in mice after administration ofacyl derivatives of uridine Time: 15 min 30 min 1 hr 2 hr Compound:Plasma Uridine Concentration (μM) Uridine 911 ± 35 415 ± 86  185 ± 215.3 ± 1.5 Triacetyl- 666 ± 70 348 ± 28  24.5 ± 7.5 15.1 ± 1.6  uridineBenzoyl- 1160 ± 45  353 ± 87  178 ± 54 8.6 ± 1.7 uridine Valeryl- 1001 ±113 347 ± 117  266 ± 1.41 7.4 ± 0.4 uridine Octanoyl-  56 ± 22 14 ± 7.4 17 ± 2.9  2.2 ± 0.04 uridine Ethoxy- 250 ± 18 83 ± 0.1  44 ± 12 2.0 ±1.6 carbonyluridine

Administration of all of the acyl derivatives of uridine results inelevated plasma uridine levels. The acyl derivatives provide sustainedformation of uridine via gradual deacylation. This may not be reflectedin plasma uridine levels, since cellular uptake of uridine can removeuridine from the circulation as it is formed by deacylation of theacylated uridine derivatives. It is important to note that the acylateduridine derivatives are generally superior to an equimolar quantity ofuridine in attenuating toxicity due to 5-FU (Table 8 in Example 3)

Example 5 Improved Therapeutic Index of 5-FU (I): TAU Rescue with 5-FUAlone in Tumor-Bearing Mice

Purpose

The purpose of this experiment was to assess and compare the ability ofuridine and TAU to increase the therapeutic index of 5-FU in atumor-bearing mouse model.

Methods

Sixty CD8F1 (BALB/C×DBA/8) female mice with first generation transplantsof CD8F1 spontaneous mammary adenocarcinoma were treated with a weeklychemotherapy regimen which included a single dose of 5-FU (150 mg/kg)followed by various rescue strategies. The average tumor size was 157 mgat the start of the chemotherapy. The weekly chemotherapy course wascompleted three times.

In order to evaluate the various rescue therapies, the animals weredivided into six groups of ten animals each as follows:

1. Saline: Saline (no 5-FU) 2. 5-FU alone: 5-FU (150 mg/kg i.p.) 3.5-FU + vehicle: 5-FU (150 mg/kg i.p.) + vehicle¹ 4. 5-FU + i.p. uridine:5-FU (150 mg/kg i.p.) + Uridine (3,500 mg/kg i.p.) 5. 5-FU + oraluridine: 5-FU (150 mg/kg i.p.) + Uridine (5,000 mg/kg p.o.)² 6. 5-FU +oral TAU: 5-FU (150 mg/kg i.p.) + TAU (7,582 mg/kg p.o.)² ¹1:1 oil-wateremulsion + 2.5% Tween/BO ²7,582 mg TAU and 5,000 mg uridine are molarequivalent doses.Results

At the conclusion of three weeks of chemotherapy, mortality in eachgroup was compared and, where a sufficient number of animals survived,body weight and tumor size were compared. The results are summarized asfollows in Table 12:

TABLE 12 Effect of combined administration of 5-FU and TAU or uridineAverage Final Group Mortality Tumor Weight (mg) 1. Saline 3/10 7,391 2.5-FU alone 9/10 * 3. 5-FU + vehicle 10/10  * 4. 5-FU + i.p. uridine 1/101,604 5. 5-FU + oral uridine 0/10  896 6. 5-FU + oral TAU 1/10 1,013*Not meaningful due to high mortality.

Mortality in the group receiving only saline was due to the progress ofthe disease, whereas mortality among the groups receiving 5-FU but norescue was due to the toxicity of 5-FU itself.

Conclusion

TAU and uridine are effective in rescuing tumor-bearing mice from thetoxic effects of 5-FU. Both agents increase the therapeutic index of5-FU in tumor-bearing mice, and allow higher doses of the drug to betolerated, with a commensurate increase in anti-cancer effect.

Example 6 Improved Therapeutic Index of 5-FU (II): TAU Rescue withCombination Chemotherapy in Tumor-Bearing Mice

Purpose

The purpose of this experiment was to assess and compare the ability ofTAU and uridine to increase the therapeutic index of 5-FU when used incombination with Phosphonacetyl-L-aspartate (PALA), methotrexate (MTX),and leucovorin (LV), in a drug dosing regimen which increases thecytotoxic potential of 5-FU.

Methods

Forty male CD8F1 mice with transplanted CD8F1 spontaneous breast tumors(initial tumor weight of 155 mg) were treated weekly with the followingregimen:

PALA--19 hr--> MTX--2.5 hr--> 5-FU--2 hr--> LV--19 hr--> LV 100mg/kg    300 mg/kg    150 mg/kg   300 mg/kg  300 mg/kg

In order to evaluate and compare the efficacy of TAU and uridine, themice were divided into four groups of ten animals each.

1. Control: Saline 2. Uridine i.p.: Uridine (3,500 mg/kg) 3. Uridine(oral): Uridine (4,000 mg/kg) 4. TAU (oral): TAU (6,066 mg/kg¹) ¹Molarequivalent of 4000 mg/kg uridine.

Saline, uridine, or TAU was administered every eight hours for a totalof 5 treatments starting two hours after each weekly dose of 5-FU. Thisweekly regimen was repeated for three successive weeks. One week afterthe third course of chemotherapy regimen, mortality in each group wasassessed and, where a sufficient number of animals survived, body weightand tumor weight were also measured.

Results

TABLE 13 Effect of TAU on mortality of tumor-bearing mice receivingcombined chemotherapy Average Final Group Mortality Tumor Weight (mg)Control 10/10  ** Uridine i.p. 0/10 110 Uridine p.o. 5/10 ** TAU p.o.1/10 162 **Not meaningful due to high mortality.

Mortality in the control group was due to toxicity of 5-FU. The tumorweight in untreated mice at this time point averages approximately 3000milligrams.

Conclusion

TAU and uridine improve the therapeutic index of 5-FU used in thisclinically, relevant combination of agents. Oral TAU was as effective asintraperitoneally-administered uridine and more effective than anequimolar dose of orally-administered uridine.

Example 7 Oral Administration of Diacetyldeoxycytidine AttenuatesHematopoietic Toxicity of Arabinosyl Cytosine

Purpose

The purpose of this experiment was to test the efficacy ofdiacetyldeoxycytidine (DAdC) and palmitoyldeoxycytidine (PdC) inameliorating the toxic effects of the chemotherapeutic agentarabinosylcytosine (Ara-C) on the hematopoietic system.

Methods

Twenty-one Balb/C female mice weighing approximately 20 grams eachreceived a daily intraperitoneal injection of Ara-C (100 mg/kg) for fivedays. These mice were divided into three treatment groups: oraladministration of water (controls); oral administration of DAdC (411mg/kg/treatment); and intraperitoneal administration of PdC (200mg/kg/treatment in 0.2% Tween 80). Mice were treated with either water,DAdC, or PdC twice daily, at 9 a.m. and 6 p.m. Treatment volume in eachcase was 0.2 ml. An additional seven mice received no Ara-C and notreatment at all (basals).

On days seven and eleven following 5-FU administration, blood (0.2-0.3ml) was collected from the seven mice in each group by retro-orbitalbleeding into EDTA for subsequent differential blood cell counting. Micewere sacrificed by cervical dislocation, and their spleens removed andweighed.

Results

Ara-C administration resulted in significantly depressed spleen weights,WBC counts, total neutrophil counts, lymphocyte counts, and plateletcounts in the control mice compared to the basal mice (Table 14). Notoxic effects of Ara-C were observed on erythropoiesis per se (RBCcounts, hemoglobin, and hematocrit).

Treatment of mice with DAdC orally and treatment with PdCintraperitoneally significantly reversed the detrimental effects ofAra-C on hematopoiesis. Spleen weight, WBC counts, total neutrophilcounts, and platelet counts were all significantly greater than those incontrol mice (Table 14).

Conclusions

Administration of DAdC or PdC to mice receiving Ara-C ameliorates thetoxic effects of Ara-C on the hematopoietic system.

TABLE 14 The effect of diacetyldeoxycytidine (DAdC) orpalmitoyldeoxycytidine (PdC) treatment on hematopoiesis in micereceiving arabinosylcytosine (Ara-C) for five days. Spleen weight is inmilligrams, WBC, neutrophil, and platelet counts are expressed as K/μl.Spleen WBC Neut PLT Basal 93.0 ± 2.5  8.66 ± .53 1.84 ± .12  854 ± 30 Control 37.1 ± 1.3  3.60 ± .55 0.04 ± .02  230 ± 21  DAdC  87.8 ± 3.5**5.00 ± .42 0.42 ± .05** 728 ± 64** PdC 126.0 ± 5.3**  5.20 ± .35* 1.22 ±.27** 327 ± 30*  *indicates p < .05 versus control **indicates p < .001versus control

Example 8 Orally Administered TAU Ameliorates the Detrimental Effects onthe Hematopoietic System of the Antiviral Chemotherapeutic AgentAzidothymidine (AZT) in Drinking Water

Purpose

The purpose of this experiment was to test the efficacy of orallyadministered triacetyluridine (TAU) in attenuating the hematopoieticdamage caused by the antiviral chemotherapeutic agent azidothymidine(AZT).

Methods

Forty-two Balb/C female mice, each weighing approximately 19 grams, weredivided into three different groups of 14 animals each. The three groupswere: basal (no AZT, no treatment), control (AZT, water), and TAU (AZT,TAU at 460 mg/kg/treatment). AZT was administered ad libitum in thedrinking water at a concentration of 1.5 mg/ml throughout the course ofthe experiment. The volume of AZT solution consumed in all treatmentgroups was similar and averaged 2.25 ml per day per mouse, resulting ina daily AZT dose of about 170 mg/kg per day. Water and TAU wereadministered in a volume of 0.2 mL three times per day for the first 24days of the study, and twice each day thereafter.

All of the animals were weighed on the day the experiment began, onceeach week, and immediately prior to sacrifice. After weighing the miceon days 24 and 35, blood (0.2-0.3 ml) was collected from seven mice ineach group by retro-orbital bleeding into EDTA for subsequentdifferential blood cell counting, including reticulocytes. Mice werethen sacrificed by cervical dislocation; their right femurs removed andthe contents expelled for cell counting; and their spleens removed andweighed.

Results

Body weights of the mice receiving AZT alone were significantly reduced(17.55±0.47 grams, p<0.002) compared to those of basal animals(19.78±0.38) at day 7, while the body weights of mice treated with TAU(19.51±0.38) were nearly identical to those of basals and significantlygreater (p<0.005) than those of controls at this same time point. Thistrend was also observed on day 13, although there were no statisticallysignificant differences between groups. Again on day 24 of thisexperiment, the body weights of the AZT controls were statisticallydepressed compared to both basals (p<0.001) and TAU-treated mice(p<0.05). At this time point the body weights of the TAU-treated animalswere also less than those of basal animals.

Day 24

The RBC counts of the mice receiving AZT only (8.49±0.16) weresignificantly reduced (p<0.01) compared to basals (9.07±0.11). The RBCcounts of those animals treated with TAU (9.01±0.09) were notsignificantly different from basals and were significantly greater(p<0.02) than those of controls.

Total neutrophil counts were also significantly reduced (p<0.02) in themice receiving only AZT (0.67±0.09) compared to basals (1.02±0.08).Neutrophil levels in those animals treated with TAU (0.91±0.06) were notsignificantly different from basals, and were significantly greater(p<0.05) than those in the AZT-only group.

Day 35

Significant detrimental effects of AZT were seen with virtually everyparameter employed at this time point. Bone marrow cellularity, WBCcounts, and lymphocyte counts were significantly depressed as were RBCcounts, hemoglobin, hematocrit compared to those from basal mice (Table15). Mean cell volume, mean cell hematocrit, and platelet counts of thecontrol mice were significantly elevated compared to those of the basalmice (Table 14). All of these parameters indicate AZT-inducedhematopoietic damage.

Treatment with oral TAU resulted in significantly higher RBC counts,hemoglobin, and hematocrit compared to AZT controls (Table 15), as wellas significantly lower mean cell volume, mean cell hematocrit, andplatelet counts compared to the control values (Table 16).

While reticulocyte number was significantly elevated in the controlgroup (0.256×10⁶ per microliter; p<0.001) versus basal counts (0.129×10⁶per microliter), those mice treated with TAU had reticulocyte levelssignificantly higher (0.371×10⁶ per microliter) than both basals(p<0.001) and controls (p<0.01).

Conclusions

From these data it is clear that orally administered TAU has beneficialeffects in animals with AZT-induced hematopoietic damage.

TABLE 15 TAU ameliorates the detrimental effects of AZT onerythropoiesis in mice. Red blood cell (RBC) counts are in M/μl,hematocrit (HCT) is expressed as a percentage, and hemoglobin (HGB) isgm/dl. RBC HCT HGB Basal 9.04 ± .08 42.52 ± .34 15.57 ± .09 Control 7.53± .13 39.29 ± .59 14.57 ± .26 TAU  8.20 ± .20**  41.26 ± .62*  15.14 ±.12* *indicates p < .05 versus control **indicates p < .01 versuscontrol

TABLE 16 TAU attenuates AZT-induced cell damage. Platelet counts (PLT)are in K/μl, mean cell volume (MCV) is in fl, and mean cell hematocrit(MCH) is measured in picograms. PLT MCV MCH Basal 736 ± 24 47.03 ± .1617.23 ± .13 Control 903 ± 18 52.16 ± .37 19.37 ± .20 TAU  809 ± 06** 50.41 ± .48*  18.49 ± .28* *indicates p < .05 versus control**indicates p < .01 versus control

Example 9 Orally Administered TAU Ameliorates the Detrimental Effects onthe Hematopoietic System of Intraperitoneally Administered AZT

Purpose

The purpose of this experiment was to test the efficacy of orallyadministered TAU in reversing the hematopoietic damage caused byparenteral administration of azidothymidine (AZT).

Methods

AZT (100 mg/kg i.p.) was given three times daily at 9 a.m., 4 p.m., andat 10 p.m. to fifty-six female Balb/C mice weighing approximately 19grams each. Three times each day, at 9 a.m., 4 p.m., and 10 p.m.,animals were treated with either water (control) or TAU at doses of 230,460, or 920 mg/kg/treatment by oral intubation. Treatment volume was 0.2ml for the control and TAU 460 groups, 0.1 ml for the TAU 230 group, and0.4 ml for the TAU 920 group. One additional group of fourteen mice wasnot given AZT or any treatments (basal).

All of the animals were weighed on the day the experiment began, on daysix and on day 13. After weighing the mice on days six and thirteenblood (0.2-0.3 ml) was collected from seven mice in each group byretro-orbital bleeding into EDTA for subsequent differential blood cellcounting, including reticulocytes. Mice were then sacrificed by cervicaldislocation; their right femurs removed and the contents expelled forcell counting; and their spleens removed and weighed.

Results

Day Six.

Bone marrow cellularity was significantly greater (p<0.05) in the grouptreated with the 230 mg/kg TAU (8.89±0.46) than in the group receivingAZT alone (7.54±0.23).

White blood cell counts were 8.23±0.38×10³ per microliter in the basalgroup. AZT reduced the WBC count to 6.8±0.66×10³ per microliter, butwhen mice were treated with TAU (920 mg/kg) in addition to receivingAZT, WBC counts were restored to basal levels (8.46±0.63×10³ permicroliter).

AZT administration resulted in a drop in RBC levels from 9.14±0.10×10⁶per microliter (basals) to 8.80±0.31×10⁶ per microliter (controls). Nosuch decrement was seen in mice receiving TAU (460 mg/kg/treatment) andAZT (9.15±0.07×10⁶ per microliter).

Day Thirteen.

For virtually every parameter employed in this study those mice givenAZT alone for 13 days showed statistically significant evidence ofhematopoietic damage. Thus, WBC counts, RBC counts, hemoglobin,hematocrit, reticulocyte counts, total neutrophil counts, and lymphocytecounts were all significantly depressed, while mean cell hematocrit andplatelet counts were significantly elevated. Concomitant treatment ofmice receiving AZT with TAU (460 mg/kg/treatment) resulted instatistically significant improvement in each and all of these measures(Table 17 and 18).

Conclusions

Concomitant treatment of mice with AZT and TAU significantly improveshematopoietic function. This is true for both the white blood cell andred blood cell indices.

TABLE 17 Oral TAU attenuates AZT-induced damage to the myelopoieticsystem in mice. These data were obtained on day 13 of the study. WBCNeutro. Lymph. Platelets K/μl K/μl K/μl K/μl Basal  6.8 ± 0.6  127 ±0.11 5.14 ± 0.33 680 ± 30 Control  5.1* ± 0.28 0.49* ± 0.07 4.02* ±0.27  1025* ± 44  TAU 7.0** ± 0.37  0.62 ± 0.03 6.06** ± 0.38  863** ±26  *= different from Basal group, P < .05 **= different from Controlgroup, P < .05

TABLE 18 Oral TAU attenuates AZT-induced damage to the erythropoieticsystem in mice. These data were obtained on day 13 of the study. RBC HGBHCT Retic. M/μl G/dL % M/μl Basal 9.16 ± 15.32 ± 43.18 ± 0.298 ± 0.060.18 0.54 0.028 Control 8.15* ± 14.08* ± 38.90* ± 0.086* ± 0.07 0.130.43 0.007 TAU 8.58** ± 14.54** ± 40.83** ± 0.162** ± 0.08 0.13 0.370.014 *= different from Basal group, P < .05 **= different from Controlgroup, P < .05

Example 10 Oral Administration of Diacetyldeoxycytidine (DAdC)Ameliorates Hematopoietic Toxicity Produced by IntraperitoneallyAdministered AZT in DBA Mice

Purpose

The purpose of this experiment was to test the efficacy of orallyadministered DAdC in reversing the hematopoietic damage caused byparenteral administration of the antiviral chemotherapeutic agentazidothymidine (AZT).

Methods

AZT (100 mg/kg i.p.) was given three times daily at 9 a.m., 4 p.m., andat 10 p.m. to twenty-eight female DBA mice weighing approximately 20grams each. Three times each day, at 9 a.m., 4 p.m., and 10 p.m.,animals were treated with either water (control) or DAdC (411mg/kg/treatment) by oral intubation. Treatment volume was 0.2 ml. Oneadditional group of fourteen mice was not given AZT or any treatment(basal).

During the first few days of treatment a total of eight mice died fromaccidents occurring during oral administration of the water and DAdC.Therefore, the number of animals in the control group and the DAdC groupwere reduced on the days of sacrifice as follows: On day 6 there werefour mice in the control group and five in the DAdC group; on day 13there were seven in the control group and three in the DAdC group. Thenumber of basal animals was seven at both time points.

On days six and thirteen blood (0.2-0.3 ml) was collected from mice ineach group by retro-orbital bleeding into EDTA for subsequentdifferential blood cell counting, including reticulocytes. The animalswere then sacrificed by cervical dislocation; their right femurs removedand the contents expelled for cell counting; and their spleens removedand weighed.

Results

Day Six

By day 6 AZT administration resulted in statistically significanthematopoietic damage, especially in those mice not receiving DAdC(controls). Thus, WBC and lymphocyte counts were significantly depressedin the controls compared to the basals, as were RBC counts, hemoglobin(HGB), hematocrit (HCT), and reticulocyte counts (Table 17). Plateletcounts were significantly elevated in these control mice. Bone marrowcell counts were also reduced 23% in the control group compared to thebasals.

In contrast, those mice receiving AZT but also treated with DAdC hadonly a slight (2.5%) reduction in bone marrow cellularity and were notstatistically different from basals. RBC counts, HGB, HCT, andreticulocyte counts in the DAdC group were not different from those inthe basal group, but were significantly greater than those in thecontrol group (Table 19).

Day Thirteen

Mice given AZT alone for 13 days showed statistically significantevidence of hematopoietic damage in nearly every category compared tobasal animals. Concomitant treatment of mice with DAdC markedlyattenuated or reversed the AZT-induced erythropoietic damage (Table 20)as was seen on day 6. Platelet counts were also significantly improvedin mice treated with DAdC (769±32; p<0.02) compared to control animals(950±34).

Conclusions

Treatment of mice receiving AZT with DAdC significantly improveshematopoietic function, especially erythropoiesis.

TABLE 19 The effect of diacetyldeoxycytidine (DAdC) treatment onerythropoiesis in mice receiving AZT for six days. RBC HGB HCTReticulocytes Basal 9.47 ± .18  13.93 ± .20  40.83 ± 0.83  .326 ± .039 Control 8.53 ± .33*  12.60 ± .40*  36.32 ± 1.54*  .017 ± .004*  DAdC9.49 ± .32** 13.96 ± .40** 40.84 ± 1.35** .205 ± .075** *indicatesdifferent from Basal, p < .05 **indicates different from Control p < .05

TABLE 20 The effect of diacetyldeoxycytidine (DAdC) treatment onerythropoiesis in mice receiving AZT for thirteen days. RBC HGB HCTReticulocytes Basal 9.95 ± .13  14.83 ± .19  42.83 ± 0.41  .637 ± .044 Control 8.51 ± .05*  13.06 ± .07*  37.21 ± 0.13*  .365 ± .022*  DAdC9.16 ± .32** 13.97 ± .39** 41.53 ± 1.17** .814 ± .069** *indicatesdifferent from Basal, p < .05 **indicates different from Control, p <.05

Example 11 Orally Administered Diacetyldeoxycytidine (DAdC) orTetrahydrouridine (THU) Ameliorate the Detrimental Effects on theHematopoietic System of Intraperitoneally Administered AZT in Balb/CMice

Purpose

The purpose of this experiment was to test the efficacy of orallyadministered DAdC or parenterally administered THU in reversing thehematopoietic damage caused by parenteral administration of theantiviral chemotherapeutic agent azidothymidine (AZT).

Methods

AZT (100 mg/kg i.p.) was given three times daily at 9 a.m., 4 p.m., and10 p.m. to twenty-one female Balb/C mice weighing approximately 20 gramseach. Three times each day, at 9 a.m., 4 p.m., and 10 p.m., sevenanimals were treated with either water (control, p.o.) or DAdC (300mg/kg/treatment, p.o.), or THU (12.5 mg/kg/treatment in 0.2% Tween 80,i.p.). Treatment volume was 0.2 ml. One group of seven mice was notgiven AZT or any treatment (basal).

On day thirteen blood (0.2-0.3 ml) was collected from mice in each groupby retro-orbital bleeding into EDTA for subsequent differential bloodcell counting, including reticulocytes. The animals were then sacrificedby cervical dislocation; their right femurs removed and the contentsexpelled for cell counting; and their spleens removed and weighed.

Results

Concomitant treatment of mice with DAdC markedly attenuated or reversedAZT-induced erythropoietic damage (Table 21). Total neutrophil countswere also significantly improved in mice treated with DAdC (1.39±0.14;p<0.01) compared to control animals (0.74±0.10).

Mice given AZT and receiving THU treatment showed significantimprovements in myelopoiesis compared to controls as well aserythropoiesis. Total white blood cell counts were significantly greaterin the THU group (6.06±0.35; p<0.05) than in the control group(4.73±0.36). Significant differences (p<0.05) were also observedcomparing the total neutrophils counts in the THU-treated group(1.16±0.15) with those of the controls (0.74±0.10). Lymphocyte countswere improved, but the differences did not reach statisticalsignificance in this experiment. In addition, reticulocyte indices(percent and M/μl) were significantly greater (p<0.05) in theTHU-treated group compared to those in the control. group.

Conclusions

Treatment of Balb/C mice receiving AZT with DAdC or THU significantlyimproves hematopoietic function.

TABLE 21 The effect of diacetyldeoxycytidine (DAdC) treatment onerythropoiesis in Balb/C mice receiving AZT for thirteen days. RBC HGBHCT Retic Basal 8.63 ± .11  15.37 ± .08  41.01 ± 0.52  .089 ± .009 Control 7.55 ± .11*  13.71 ± .22*  35.96 ± 0.46*  .039 ± .004*  DAdC8.26 ± .13** 14.97 ± .15** 39.51 ± 0.61** .059 ± .006** *indicatesdifferent from Basal, p < .05 **indicates different from Control, p <.05

Example 12 Plasma Uridine Levels after Oral Administration of Uridine orTriacetyluridine (TAU), with or without Dipyridamole

Purpose

The purpose of this experiment was to demonstrate that TAU is a moreeffective orally-active agent for elevating plasma uridine levels thanis uridine itself, and furthermore to demonstrate the effect ofdipyridamole (DPM), a nucleoside uptake-blocker which also has antiviralactivity, on plasma uridine levels after administration of TAU oruridine.

Methods

Female Balb/C mice with a body weight of 20 grams were divided into fourgroups of 8 animals each:

1. Uridine (1000 mg/kg) p.o.

2. TAU (1500 mg/kg) p.o.

3. Uridine (1000 mg/kg) p.o.+DPM (25 mg/kg i.p.)

4. TAU (1500 mg/kg) p.o.+DPM (25 mg/kg i.p.)

(1500 mg/kg TAU is the molar equivalent of 1000 mg/kg uridine)

Dipyridamole was administered by intraperitoneal injection 30 minutesprior to uridine or TAU

Uridine was administered orally by gavage as an aqueous solution in avolume of 0.4 ml.

TAU was administered by gavage in an emulsion vehicle (1:1 cornoil/water with 2.5% Tween 80).

Two mice from each group were bled from the suborbital plexus at eachtime point: 0 (Basal uridine levels before TAU or uridineadministration), 0.5, 1, 2, and 4 hours after administration of TAU oruridine

Plasma samples (0.1 ml) were deproteinized by addition of 0.2 mlmethanol followed by centrifugation. Samples were lyophilized and thenreconstituted with HPLC buffer (100 mM ammonium acetate, pH 6.5, forsubsequent assay of uridine by reverse phase HPLC with UV absorbancedetection (254 nm).

Data points are the mean of two samples for each time point.

Results

After oral administration of uridine, plasma uridine reached peakconcentrations of 6 micromolar. In contrast, oral administration of anequimolar dose of TAU resulted in peak plasma uridine levels of 260micromolar, thus demonstrating the marked advantage of TAU over uridineas an orally active means of elevating plasma uridine levels.

Dipyridamole further enhanced (approximately two-fold) the amplitude(peak uridine levels of 460 micromolar) and duration of blood uridinelevels after oral administration of TAU.

Dipyridamole similarly improved blood uridine level maintenance afteroral uridine, although levels were much lower than in the correspondingmice that received TAU (peak plasma uridine levels of 20 micromolar).

These results are summarized in Table 22.

TABLE 22 Plasma uridine concentrations after oral administration ofuridine or TAU, with or without dipyridamole. Plasma uridine levels (μM)after TAU or uridine administration Treatment 0 hr 0.5 hr 1 hr 2 hr 4 hrUridine 2 4 6 4 2 TAU 2 240 260 110 12 Uridine + DPM 2 10 20 10 4 TAU +DPM 2 280 460 440 32Conclusions

Plasma uridine levels after oral administration of TAU were much higherthan were observed after oral administration of uridine. TAU is thus amuch better source of plasma uridine after oral administration than isuridine itself. Dipyridamole inhibits uridine uptake into some celltypes, and thereby enhances the amplitude and duration of plasma uridineconcentrations after administration of TAU or uridine.

Example 13 Modulation of Toxicity of Oral Tegafur with TAU and Uracil

Purpose:

Tegafur (5-fluoro-1-(tetrahydro-2-furfuryl)uracil) is an orally activeprodrug of 5-fluorouracil; it is enzymatically converted to 5-FU duringand after absorption from the intestinal tract into the bloodstream. Fortreatment of cancer, Tegafur, is typically administered orally in aformulation also containing uracil, in a 1:4 molar ratio of Tegafur touracil; tegafur formulated with uracil is currently used clinically inhumans. Uracil potentiates the activity of the 5-FU formed from Tegafurby competitively inhibiting dihydrouracil dehydrogenase, an enzyme whichdegrades 5-FU.

After administration of high doses of tegafur+uracil, mice lose asubstantial amount of body weight, indicating gastrointestinal toxicity.After oral administration, it is believed that uracil potentiates thelocal toxicity of 5-FU formed in intestinal cells during passage ofTegafur into the bloodstream. It is therefore desirable to utilize withtegafur, or other orally active prodrugs of 5-FU, an agent whichinhibits breakdown of 5-FU (or otherwise potentiates its activity)primarily in the circulation (after absorption) rather than locally inthe gut.

Triacetyluridine (TAU), like other acyl derivatives of uridine andcytidine of the invention, is converted to uridine and uracil during andafter absorption into the bloodstream; when present at the same time as5-FU, both uridine and uracil are capable of potentiating 5-FUcytotoxicity. Therefore, the cytotoxicity of oral Tegafur+uracil versusTegafur+TAU was assessed. Blood cell counts were utilized as an index ofcytotoxicity of 5-FU in the circulation (and by extension, of antitumorpotency). Body weight loss was used as an index of gastrointestinaltoxicity.

Methods:

Three groups of mice received Tegafur by oral intubation in a dose of400 mg/kg per mouse. The initial body weight of mice in each group was19.0±0.6 grams. One of these groups also received uracil in a molarratio of 4:1 to the tegafur dose, and another group received TAU, alsoin a 4:1 molar ratio to Tegafur. Body weights were monitored. Six daysafter Tegafur administration, blood samples were taken for differentialcell counts.

Results:

Tegafur alone at a dose of 400 mg/kg produced a significant drop inneutrophil numbers, but did not significantly affect platelet counts;body weight was reduced only slightly (7%) compared to untreated (basal)animals. Tegafur plus uracil produced a more severe drop in neutrophilcounts than did Tegafer alone, and also reduced platelet counts andcaused a substantial (29%) loss of body weight. Tegafur plus TAUproduced blood cell count changes similar to those found after tegafurplus uracil, but did not cause a change in body weight. The blood cellcounts found after oral administration of either Tegafur+TAU orTegafur+uracil are similar to those observed after a therapeuticallyeffective dose of 5-fluorouracil (e.g. 150 mg/kg) administeredsystemically. However, the weight loss caused by Tegafur+uracil isunacceptable in the context of cancer chemotherapy. The excellentsystemic cytotoxicity of oral Tegafur+TAU (better than Tegafur alone andat least equivalent to Tegafur+uracil) and the absence of a loss in bodyweight (especially in contrast to the marked weight loss in animalsreceiving Tegafur+TAU) indicate that TAU is useful in potentiatingsystemic cytotoxicity (and therefore antitumor potency) of orally-activefluorouracil prodrugs without a proportional increase ingastrointestinal toxicity. Combination of an orally active fluorouracilprodrug with an acylated non-methylated pyrimidine nucleoside derivativetherefore permits better oral delivery of therapeutically effectiveamounts of the important antineoplastic drug fluorouracil than isobtained with current methods and compositions.

TABLE 23 Uracil vs TAU for enhancing cytotoxicity of Tegafur Body weightNeutrophils Platelets (grams) (K/μl) (K/μl) Basal 19.6 ± 0.5 2.60 ±0.510  984 ± 25  Tegafur-400 18.2 ± 0.7 0.40 ± 0.085* 956 ± 30 Tegafur-400 +  14.0 ± 0.3* 0.02 ± 0.002* 448 ± 31* Uracil FT-400 + 19.8± 0.4 0.02 ± 0.003* 334 ± 33* TAU *Different from Basal values, P < .01

All drugs were administered orally in a single dose. Blood samples weretaken for cell counts 6 days after drug administration; body weightswere also recorded at this time.

Example 14 Antitumor Efficacy of Tegafur and TAU vs Tegafur and Uracil

Purpose

Tegafur (FT) is an orally active prodrug of fluorouracil. Uracil, at anoptimum molar ratio of 4:1, potentiates the antitumor efficacy of FT.The dose limiting toxicity of the FT-uracil combination is damage to theintestinal mucosa. Increasing doses of FT-uracil also result inhematopoietic damage.

The purpose of the present study was to compare the antitumor efficacyof FT in combination with either uracil or triacetyluridine (TAU) inrats bearing the Walker 256 carcinosarcoma. The essential questionaddressed in this experiment was whether or not FT+TAU inhibits tumorgrowth as well as FT+uracil, while causing less intestinal toxicity(body weight changes). Blood cell damage was also assessed and compared.

Methods

Animals

Male Sprague-Dawley rats with an initial body weight of approximately120 grams.

Tumors

Pooled ascites fluid containing Walker 256 cells at a density of2.47×10⁸ cells/milliliter was collected from three donor rats. Analiquot containing 4.94×10⁷ cells was injected subcutaneously in theright flank of each rat in the anti-tumor efficacy study. This resultedin formation of solid subcutaneous tumors.

Treatment

FT was administered at doses of 40, 60, and 80 mg/kg/day in a 1:4 molarratio with either uracil or TAU. The vehicle used was 1%hydroxypropylmethylcellulose. Treatment was initiated (day 1) five daysafter tumor implantation. Vehicle and vehicle plus drugs wereadministered orally by gavage each day for seven days (day 1 through 7)in a volume of 1.2 milliliters per 100 grams of body weight. Tumor sizewas measured in situ on day 8. On day 10 blood samples were obtainedand, after sacrifice of the animals, tumor size and weight weredetermined.

Treatment Groups

1. Vehicle (control)

2. FT 40 mg/kg+uracil

3. FT 60 mg/kg+uracil

4. FT 80 mg/kg+uracil

5. FT 40 mg/kg+TAU

6. FT 60 mg/kg+TAU

7. FT 80 mg/kg+TAU

n=7 animals/group

Measurements

Body weights were determined prior to treatment on days 1, 3, 5, and 7.On day 10 the animals were weighed prior to sampling and sacrifice. Bodyweights are expressed as the percent of body weight change over thecourse of the experiment.

Tumor size was measured in situ on day 8 and the tumor volume was thencalculated using the following formula:length×width²/2Following sacrifice on day 10 the tumor was exposed, the size measuredin situ, and the tumor volume calculated using the formula above. Thetumor was then removed and the tumor weight determined. Tumor data arealso expressed as T/C %. T/C % is:mean tumor size in drug-treated rats×100 mean tumor size in control rats

Complete blood cell counts with differential were determined using bloodsamples obtained by cardiac puncture immediately prior to sacrifice onday 10.

Results

The effects of FT+uracil and FT+TAU on tumors and on body weights asassessed on day 8 are summarized in Table 24. The data show that FT+TAUhas greater anti-tumor efficacy and preserves body weight better thanequivalent doses of FT+uracil at each dose of FT. For example, at the FT60 dose the tumor volume and T/C percent for the FT-TAU treated groupare less than half those of the uracil-treated group. At that same doselevel (FT 60) the body weight change is 52.1% in the FT-TAU groupcompared to 7.8% in the FT-uracil group. Higher doses of FT are moreeffective in preventing tumor growth than lower doses.

Tumor and body weight data obtained on day 10 are presented in Table 25.Tumor values—T/C percent, tumor volume and tumor weight—aresignificantly lower and body weight gains significantly greater in therats treated with FT-TAU than in those treated with FT-uracil.

The hematopoietic effects of FT treatment are recorded in Table 26.Platelet counts are preserved at all FT dose levels in the FT-TAUtreated animals, while dropping precipitously at increasing doses of FTin the FT-uracil groups. Total white, blood cell counts and lymphocytesare also maintained better in the FT-TAU groups at the higher, moreeffective doses of FT. At the FT 40 dose, neutrophil counts are lessseverely attenuated in the FT-TAU group than in the equivalent FT-uracilgroup. In fact, at each FT-TAU dose level, neutrophil counts areapproximately twice those observed in the corresponding FT-uracil group.

The results of this experiment indicate that the use of oral TAU incombination with FT has significant advantages over the FT-uracilcombination. FT-TAU has greater anti-tumor activity than equivalentdoses of FT-uracil while causing less intestinal and hematopoieticdamage.

TABLE 24 Antitumor effects of FT with uracil or TAU in rats bearingWalker 256 Carcinosarcoma: Day 8 Group Tumor volume T/C Body wt. change(mm³ ± SE) (%) (%) Control 3098 ± 372 100.0 100.0 FT 40 + Uracil 1651 ±135 53.3 51.0 FT 60 + Uracil 1051 ± 123 30.8 −1.7 FT 80 + Uracil  848 ±124 27.4 −26.0 FT 40 + TAU 1244 ± 209 40.2 78.0 FT 60 + TAU 1006 ± 11432.5 52.0 FT 80 + TAU 557 ± 49 18.0 23.7 FT dose = mg/kg/day for 7 daysFT/Uracil = 1:4 molar ratio FT/TAU = 1:4 molar ratio

TABLE 25 Antitumor effects of FT with uracil or TAU in rats bearingWalker 256 Carcinosarcoma: Day 10 Group Tumor volume T/C Body wt. change(g ± SE) (%) (%) Control 4.16 ± 0.55 100.0 100.0 FT 40 + Uracil 3.62 ±0.35 87.0 62.2 FT 60 + Uracil 2.80 ± 0.28 67.3 39.0 FT 80 + Uracil 1.50± 0.58 36.1 −7.5 FT 40 + TAU 2.14 ± 0.31 51.4 93.2 FT 60 + TAU 2.18 ±0.42 52.4 58.9 FT 80 + TAU 1.77 ± 0.25 42.5 30.3 FT dose = mg/kg/day for7 days FT/Uracil = 1:4 molar ratio FT/TAU = 1:4 molar ratio

TABLE 26 Blood cell counts after FT + uracil and FT + TAU: Day 10 WBCNeutrophils Lymphocytes Platelets Control 11.8 ± 0.8  1.84 ± .24 9.9 ±0.8 844 ± 44  FT 40 + Uracil 9.7 ± 1.8 0.68 ± .18 8.5 ± 1.5 866 ± 117FT-60 + Uracil 5.9 ± 1.1 0.27 ± .11 5.6 ± 1.0 602 ± 147 FT 80 + Uracil3.6 ± 1.0 0.14 ± .12 4.3 ± 1.1 171 ± 49  FT 40 + TAU 8.2 ± 0.9 0.91 ±.21 7.0 ± 0.8 913 ± 117 FT 60 + TAU 5.3 ± 0.9 0.27 ± .05 5.0 ± 0.9 830 ±83  FT 80 + TAU 5.6 ± 0.8 0.12 ± .07 5.4 ± 0.7 534 ± 136 All blood cellcount units are K/μl FT dose = mg/kg/day for 7 days FT/Uracil = 1:4molar ratio FT/PN401 = 1:4 molar ratio

Example 15 Synthesis of Ethoxycarbonyluridine

To an ice cold solution of 0.5 grams (1.76 millimoles) grams of2′,3′-isopropylidene uridine in 10 ml pyridine, 2.64 mmoles (1.5equivalents) of ethylchloroformate was added dropwise while stirring.The reaction was allowed to warm up to room temperature (25° C.) andstirred overnight (18 hours), at which point TLC (9:1chloroform/methanol) showed complete conversion of starting material toa single product. The solvent was removed by rotary evaporation underhigh vacuum, giving a light beige syrup which was carried over into thesubsequent deprotection step.

The syrup was dissolved in 15 ml of 50% formic acid and heated at 60-70°C. for two hours, at which point TLC showed quantitative removal of theisopropylidene group. Water and formic acid were removed by evaporationunder high vacuum, giving a light beige-pink syrup which was applied toa silica gel column and eluted with 95:5 chloroform/methanol. Fractionscontaining the product were collected, pooled, and evaporated, giving afaintly pink glassy product.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the invention.

1. A method for treating toxicity due to a pyrimidine nucleoside analogin an animal comprising administering to said animal a pharmaceuticallyeffective amount of an acylated derivative of uridine or cytidineselected from the group consisting of triacetyluridine andethoxycarbonyluridine or triacetylcytidine, wherein said pyrimidinenucleoside analog is selected from the group consisting of5-fluorouracil (5-FU), Tegafur, 5-fluoroorotate,5′-deoxy-5-fluorouridine, 5-fluorouridine, 2′-deoxy-5-fluorouridine,fluorocytosine, trifluoromethyl-2′-deoxyuridine, arabinosyl cytosine,cyclocytidine, 5-aza-2′-deoxycytidine, arabinosyl 5-azacytosine,6-azacytidine, N-phosphonoacetyl-L-aspartic acid (PALA), pyrazofurin,6-azauridine, azaribine, thymidine, 3-deazauridine, AZT,dideoxycytidine, 5-ethyl-2′-deoxyuridine, 5-iodo-2′deoxyuridine,5-bromo-2′-deoxyuridine, 5-methylamino-2′-deoxyuridine,arabinosyluracil, dideoxyuridine and(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine; and wherein saidtoxicity is selected from the group consisting of damage tohematopoietic tissue and damage to mucosal tissues.
 2. A method as inclaim 1 wherein said toxicity is damage to hematopoietic tissue.
 3. Amethod as in claim 1 wherein said toxicity is damage to mucosal tissues.4. A method as in claim 1 wherein said administering step also includesadministering an inhibitor of uridine phosphorylase selected from thegroup consisting of benzylacyclouridine, benzyloxybenzylacyclo-uridine,aminomethyl-benzylacyclouridine,aminomethyl-benzyloxybenzylacyclo-uridine,hydroxymethyl-benzylacyclouridine,hydroxymethyl-benzyloxybenzylacyclouridine, 2,2′-anhydro-5-ethyluridine,5-benzyl barbiturate, 5-benzyloxybenzyl barbiturate,5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate,5-benzyloxybenzylacetyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate, and5-methoxybenzylacetylacyclobarbiturate.
 5. A method as in claim 1wherein said acylated derivative is triacetylcytidine, and saidadministering step also includes administering an inhibitor of cytidinedeaminase selected from the group consisting of tetrahydrouridine andtetrahydro-2′-deoxyuridine.
 6. A method as in claim 1 wherein saidadministering step also includes administering an inhibitor ofnucleoside transport selected from the group consisting of dipyridamole,probenicid, lidoflazine and nitrobenzylthioino sine.
 7. A method as inclaim 1 wherein said administering step also includes administering anagent which enhances hematopoiesis selected from the group consisting ofIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, granulocyte colonystimulating factor, granulocyte/macrophage colony stimulating factor,stem cell factor, erythropoietin, glucan, and polyinosine-polycytidine.8. A method as in claim 1 wherein said administering step also includesadministering a compound capable of enhancing the uptake andphosphorylation of nucleosides into cells selected from the groupconsisting of insulin and insulinogenic carbohydrate.
 9. A method as inclaim 1, wherein said acylated derivative of uridine istriacetyluridine.
 10. A method as in claim 9, wherein said pyrimidinenucleoside analog is 5-fluorouracil (5-FU).
 11. A method as in claim 9,wherein said pyrimidine nucleoside analog is AZT and said toxicity isdamage to hematopoietic tissue.